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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Received for publication
January 15, 2003
Accepted
April 8, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Pardridge, 1999;
Lee et al., 2001). The
molecular basis of the barrier function involves highly developed tight
junctions between the BCEC, metabolic enzymes, and efflux transporters located
on the plasma membrane of the BCEC. Due to the tight junctions, it is unlikely
that exogenous or endogenous compounds reach the brain via a paracellular
route. Thus, the transcellular route is the major pathway for the exchange of
compounds between the brain parenchyma and the circulating blood.
It has been shown that there is a positive correlation between the
lipophilicity and the permeability coefficient across the BBB
(Levin, 1980). Thus, certain
drugs with high lipophilicity can pass into the brain through the BBB. There
are some drugs that achieve a lower brain distribution than expected from
their lipophilicity, however. This has been explained by the efflux transport
at the BBB. Indeed, anti-tumor agents such as doxorubicin and vincristine
exhibit poor brain penetration despite their high lipophilicity
(Levin, 1980). The
unexpectedly low permeability of these drugs across the BBB can be accounted
for by P-glycoprotein-mediated efflux at the BBB.
Employing a microinjection technique (BEI method), the efflux transport
systems for organic anions at the BBB have been investigated. It has been
shown that transporters are involved in the efflux of amphipathic organic
anions such as taurocholic acid and conjugated steroids such as
17
-estradiol-D-17-glucuronide (E217G),
estrone-3-sulfate (E1S), and dehydroepiandrosterone sulfate, and a
hydrophilic organic anion, PAH, across the BBB
(Kitazawa et al., 1998;
Asaba et al., 2000;
Hosoya et al., 2000;
Sugiyama et al., 2001). A
member of the hepatic organic anion transporter family, organic anion
transporting polypeptide (Oatp) 2 has been identified on both the luminal and
abluminal membrane of the BCEC (Gao et al.,
1999). Since Oatp2 accepts amphipathic organic anions described
above as substrates (Reichel et al.,
1999), it has been a candidate transporter responsible for the
elimination of amphipathic organic anions from the brain. However, PAH is not
a substrate of Oatp2 (Reichel et al.,
1999), suggesting the involvement of additional organic anion
transporter(s) in the extrusion of PAH from the cerebrum across the BBB.
Since PAH is a typical substrate of the Oat family, which consist of three
members in rats (rOat1 to rOat3) (Inui et
al., 2000; Sekine et al.,
2000; Dresser et al.,
2001), the efflux transport of PAH across the BBB may be mediated
by the member(s) of this family. Among the Oat family in rats, the expression
of rOat1 and rOat3 in the brain has been reported. rOat1 has been isolated
from rat kidney by the expression cloning method
(Sekine et al., 1997).
Functional characterization has shown that the properties of rOat1 are
consistent with those of the classical organic anion/dicarboxylate exchanger
responsible for multispecific organic anion transport at the basolateral
membrane of renal proximal tubules
(Shimada et al., 1987).
Northern blot analysis revealed that rOat1 is expressed predominantly in the
kidney and weakly in the brain (Sekine et
al., 1997). rOat3 was first isolated from the brain using the
RT-PCR cloning method, and Northern blot analysis revealed that rOat3 mRNA is
expressed in the liver, brain, kidney, and weakly in the eye
(Kusuhara et al., 1999). rOat3
accepts amphipathic organic anions such as E217G and
E1S as well as PAH as its substrates
(Kusuhara et al., 1999;
Sugiyama et al., 2001). In the
brain, rOat3 has been shown to be expressed in the choroid plexus
(Nagata et al., 2002) as well
as in the brain capillaries (Ohtsuki et
al., 2002). In the choroid plexus, rOat3 is responsible for the
uptake of PAH and PCG from the cerebrospinal fluid
(Nagata et al., 2002). This
information suggests the possibility that rOat1 and rOat3 in the brain may be
responsible for the elimination of PAH from the cerebrum.
In the present study, the expression of rOat1, rOat2, and rOat3 at the BBB was investigated using RT-PCR analysis, Western blotting, and immunohistochemistry. The contribution of rOat3 to the efflux of organic anions such as PAH and PCG from the brain into the blood circulation was evaluated using the BEI method.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Animals
Sprague-Dawley male rats (supplied by Japan SLC, Shizuoka Ken, Japan)
weighing 220 to 250 g were used throughout this study and had free access to
food and water.
Capillary Isolation
The brain capillary-enriched fraction was isolated from rat cerebrum, as
described previously with some modifications
(Boado and Pardridge, 1991).
Briefly, pieces of gray matter were gently homogenized in 3 volumes (v/w) of
buffer A (103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 15 mM HEPES, pH 7.4),
and then the homogenate was centrifuged (5800 g) after adding dextran (15%).
The resulting pellet was resuspended in buffer B [103 mM NaCl, 4.7 mM KCl, 2.5
mM CaCl2, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 15 mM HEPES, 25 mM NaHCO3, 10 mM
D-glucose, 1 mM Na+-Pyruvate, 0.5% (w/v) BSA, pH 7.4]
and filtered through a 200-µm nylon mesh. The filtrate was passed over a
column of glass beads and washed with 500 ml of buffer B. The capillaries
adhering to the beads were collected by gentle agitation followed by
centrifugation at 500 g. All steps in the isolation procedure were carried out
at 4°C in pregassed (95% O2/5% CO2) solutions.
RT-PCR Analysis
Total RNA from the rat brain capillary-enriched fraction, kidney, and liver
was prepared by a single-step guanidinium thiocyanate procedure using Isogen
(Nippon Gene Co., Ltd., Toyama, Japan). The RNA was then reverse-transcribed
using a random-hexamer primer (Takara, Kyoto, Japan). cDNA fragments were
amplified by PCR with the cDNA prepared from the brain capillary-enriched
fraction, kidney, and liver as a template using Ready-To-Go PCR Beads
(Amersham Biosciences, Inc., Piscataway, NJ). The following primers were used
to amplify rOat1, rOat2, and rOat3 cDNA: forward primer,
5'-cttgaactacctgcagacag-3' and reverse primer,
5'-atggagagacagaggaagag-3' for rOat1, forward primer,
5'-agacctgttccgaacatctc-3' and reverse primer,
5'-gagtgcagtaagtcccaatc-3' for rOat2, and forward primer,
5'-gacctggagttatctcatgc-3' and reverse primer,
5'-ccggaacaagtcagataagc-3' for rOat3. PCR was performed according
to the following procedure: 96°C for 30 s, 55°C for 30 s, and 72°C
for 1 min (40 cycles). The sequences of the amplified fragments were confirmed
by DNA sequencing using a DNA sequencer (model 377 DNA sequencer; PerkinElmer
Instruments, Norwalk, CT).
Antiserum
Anti-rOat3 antiserum was raised in rabbits against a synthetic peptide
consisting of the 16 carboxyl-terminal amino acids of rOat3 coupled to keyhole
limpet hemocyanin at its carboxyl-terminal via an additional tyrosine.
Western Blotting
The brain capillaries were resuspended in ice-cold 1% Triton-100/PBS (500
µl for 10 mg of capillary proteins) and stored at 4°C for 20 min. The
sample was centrifuged at 15,000g for 15 min, and the supernatant was
used for Western blotting. The rat kidney plasma membrane fraction was
prepared by the standard procedure
(Nakajima et al., 2000). Brain
capillary proteins (10 µg) and rat kidney plasma membrane proteins (1
µg) were electrophoresed on 10% SDS-polyacrylamide gel with a 4.4% stacking
gel. Separated proteins were transferred to a polyvinylidene diflouride
membrane using a blotter at 15 V for 1 h. The membrane was blocked with
Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% skimmed milk for
1 h at room temperature. After washing three times with TBS-T for 5 min, the
membrane was incubated with anti-rOat3 antiserum (1:500 dilution) at room
temperature for 1 h. After washing, the membrane was allowed to bind a
horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham Biosciences
UK, Ltd.) diluted 1:5000 in TBS-T for1hat room temperature and detected using
ECL plus (Amersham Biosciences UK, Ltd.).
Deglycosylation of rOat3
N-Linked carbohydrate groups were cleaved from the rOat3 protein in the
brain capillaries and the kidney plasma membrane using PNGase F
(N-glycosidase F) (New England Biolabs, Inc., Beverly, MA). Aliquots
of 10 µg of capillary proteins or 3 µg of kidney proteins were added to
a 10-µl volume of 50 mM sodium phosphate buffer (pH 7.5) containing 1%
NP-40 and 200 units/µl N-glycosidase F, with a final SDS
concentration of 0.5%. As a control, distilled water was substituted for
N-glycosidase F in an otherwise identical reaction mixture. After 1 h
incubation at 37°C, the samples were subjected to Western blotting, as
described above.
Immunohistochemical Staining
Double Immunostaining with Antibodies to rOat3 and PGlycoprotein. A
7-week-old male Sprague-Dawley rat was perfused with PBS for 5 min, followed
by 2% paraformaldehyde for 5 min. Then, the brain was removed and stored in 2%
paraformaldehyde for 1 h at 4°C. Before sectioning, the brain was infused
with 18% sucrose. Cryostat sections (10 µm in thickness) were fixed in
methanol at –20°C for 10 min, washed three times with PBS, and
blocked with 1% BSA/PBS at room temperature for 15 min. Double staining was
performed incubating the brain slices with anti-rOat3 antiserum (1:100
dilution in 1% BSA/PBS) and C219 (1:40 dilution in 1% BSA/PBS) at 4°C
overnight. After washing with PBS, sections were incubated with secondary
antibodies (Alexa Fluor 488 anti-rabbit IgG and Alexa Fluor 568 anti-mouse
IgG, diluted to 1:100; Molecular Probes, Eugene, OR) for 1 h and mounted in
Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
Double Immunostaining with Antibodies to rOat3 and Glial Fibrillary Acidic Protein (GFAP). Cryostat sections were prepared as described above. To activate the antigen, sections were treated with microwaves for 5 min in 10 mM citric acid buffer, pH 6.0. Then, sections were fixed in ethanol at 4°C for 30 min, followed by acetone at room temperature for 1 min. After washing with PBS, sections were incubated with 0.5% Triton-X/PBS at room temperature for 15 min, washed again, and blocked with 1% BSA/PBS for 15 min. Double-staining was performed as described above using anti-rOat3 antiserum (1:100) and monoclonal mouse anti-human GFAP (DAKO Japan, Kyoto, Japan) (1:25) both diluted in 1% BSA, 0.2% Triton X-100/PBS at 4°C overnight. After washing with PBS, sections were incubated with secondary antibodies (Alexa Fluor 488 anti-rabbit IgG and Alexa Fluor 568 anti-mouse IgG, diluted to 1:100) for 1 h and mounted in Vectashield mounting medium.
BBB Efflux Study
The efflux of test compounds from the brain after microinjection into the
cerebral cortex was investigated using the BEI method, as described previously
in Kakee et al. (1996).
Briefly, rats were anesthetized with intramuscular doses of ketamine (125
mg/kg) and xylazine (1.22 mg/kg) and placed in a stereotaxic frame (Narishige
Co., Tokyo, Japan). After exposure of the skull, a small hole was made in the
scalp, 0.2 mm anterior and 5.5 mm lateral to the bregma, and the
microinjection needle was inserted through the hole to a depth of 4.5 mm (Par2
region). [3H]PAH (0.125 µCi/rat) or [3H]PCG (0.125
µCi/rat) with [14C]carboxyl-inulin (2.5 nCi/rat), which is a
nonpermeable reference compound, dissolved in 0.5 µl ECF buffer (122 mM
NaCl, 25 mM NaHCO3, 10 mM D-glucose, 3 mM KCl, 1.4 mM
CaCl2, 1.2 mM MgSO4, 0.4 mM
K2HPO4, and 10 mM HEPES, pH 7.4) in the presence or the
absence of different concentrations of various inhibitors was injected into
the Par2 region. The actual concentrations of labeled compounds in the
injectate were 55 µM, 13 µM, and approximately 380 µM for
[3H]PAH, [3H]PCG, and [14C]carboxyl-inulin,
respectively. After intracerebral injection of the test compounds, rats were
decapitated, and the left and right cerebrum was removed at appropriate times.
The excised cerebrum was dissolved in 2.5 ml of 2N NaOH at 55°C overnight,
and the radioactivity in the brain specimens was measured in a liquid
scintillation counter (LS 6000SE; Beckman Coulter, Inc., Fullerton, CA) after
adding 14 ml of scintillation fluid (Hionic-fluor; Packard Instruments,
Meriden, CT). The 100-BEI (percentage) that represents the remaining
percentage of the test compounds in the cerebrum is described by the following
equation: 100-BEI (%) = [(amount of test drug in the brain/amount of reference
in the brain)/(amount of test drug injected/amount of reference injected)]
x 100. The elimination rate constant of the compounds from the brain
(kel) was obtained by fitting the 100-BEI (%) versus time
data. A nonlinear least-squares regression program (MULTI)
(Yamaoka et al., 1981) was
used for the calculation.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-actin fragments. DNA sequencing of the amplified products was identical
to rOat1, rOat2, and rOat3, respectively.
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The expression of rOat3 in brain capillaries was also confirmed at the protein level by Western blot analysis (Fig. 1B). An antiserum against rOat3 recognized approximately 75- and 65-kDa proteins in the brain capillary-enriched fraction and kidney plasma membrane fraction, respectively (lanes 1 and 2). These bands were abolished when preabsorbed anti-serum for rOat3 was used (lanes 3 and 4), suggesting that the positive bands were specific for the antigen peptide.
To examine the degree of glycosylation of rOat3 in the brain capillary-enriched fraction and kidney, protein samples were deglycosylated with N-glycosidase F and subsequently subjected to Western blot analysis (Fig. 1C). The resulting bands show the same molecular weight for the brain capillary-enriched fraction and kidney plasma membrane fraction (lanes 3 and 4), suggesting that the difference in the molecular weight is due to the difference in the degree of glycosylation between the brain capillaries and the kidney.
Immunohistochemical Staining of rOat3. The localization of rOat3 at
the BBB was examined by immunohistochemical staining using frozen sections of
rat brain. Positive staining signals were observed around the brain
microvessels for rOat3 (green) and P-gp (red)
(Fig. 2A), and the intensity of
each signal was plotted along a cross-section of the microvessel
(Fig. 2B). rOat3 produced a
wider signal across the microvessel than P-gp, which is a luminal marker
protein (Tsuji et al., 1992).
Furthermore, the signal for rOat3 (green) did not overlap with that for GFAP
(red), a marker for astrocytes, indicating that rOat3 is not expressed in the
astrocytes but in the BCEC (Fig.
2C). These profiles strongly suggest that rOat3 is located on the
abluminal membrane of the BCEC. The signal for rOat3 overlapped that for P-gp
to some extent, indicating that rOat3 may be localized on the luminal as well
as the abluminal membrane of the BCEC. Such characteristic immunostaining did
not appear when normal rabbit serum was used as a negative control
(Fig. 2D; red/P-gp).
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Time Profile of the Efflux of [3H]PAH and [3H]PCG from the Brain across the BBB. The time profile of the remaining percentage of PAH or PCG in the cerebrum after intracerebral injection is shown in Fig. 3. Approximately 40 and 60% of the administered dose of PAH and PCG was eliminated from the cerebrum into the systemic circulation within 20 min, respectively. The apparent elimination rate constant (kel) was determined as 0.039 ± 0.004 min–1 for PAH and 0.043 ± 0.006 min–1 for PCG.
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Concentration-Dependent Efflux of PAH and PCG from the Brain and Mutual
Inhibitory Effects. The apparent elimination rate constant
(kel) of PAH or PCG was reduced in a dose-dependent manner
(Fig. 4, A and B). Taking the
dilution factor of 45 in the cerebrum into consideration
(Kakee et al., 1996), the
Michaelis-Menten constant, Km, for the efflux of PAH and
PCG across the BBB was 168 ± 84 and 28.7 ± 4.5 µM,
respectively (Table 1).
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The efflux of [3H]PAH or [3H]PCG from the brain was inhibited by unlabeled PCG or unlabeled PAH, respectively, in a concentration-dependent manner (Fig. 4, C and D). According to the kinetic analyses, the Ki values of PAH and PCG for the elimination of PCG and PAH from the cerebrum across the BBB were 106 ± 36 and 58.1 ± 15.8 µM, respectively (Table 1).
Effect of Cimetidine and Pravastatin on the Efflux of [3H]PAH
and [3H]PCG from the Brain Parenchyma. Cimetidine, which is not
only a typical substrate of the organic cation transporter family
(Dresser et al., 2001), but
also a substrate of rOat3 (Nagata et al.,
2002), and pravastatin, which is a substrate of rOat3
(Hasegawa et al., 2002), were
used as inhibitors to confirm the involvement of rOat3 in the elimination of
[3H]PAH and [3H]PCG from the cerebrum. As shown in
Fig. 5, A and B, the inhibitory
effects of cimetidine and pravastatin were dose-dependent and statistically
significant.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). In
contrast, no expression of rOat1 or rOat2 at the BBB was detected in this
study. Therefore, we conclude that, among the rat Oat family, only rOat3 is
expressed at the BBB. The existence of rOat3 in the BCEC was further confirmed
by Western blot analysis (Fig.
1B). The molecular weight of the positive signal detected in the
brain capillary-enriched fraction was greater than that in the kidney plasma
membrane fraction. This difference in the molecular weight was shown to be due
to the difference in the degree of glycosylation between the brain capillaries
and kidney (Fig. 1C). There is
one additional band around 48 kDa in the brain capillary-enriched fraction.
This may represent a degradation product of rOat3 arising during the isolation
of the brain capillaries since this band was abolished when preabsorbed
antiserum for rOat3 was used. Immunohistochemical staining demonstrated that
rOat3 is located on the abluminal membrane of the BCEC
(Fig. 2). It is possible that
rOat3 is expressed on the luminal as well as the abluminal membrane of the
BCEC since the signal for rOat3 overlapped that for P-gp to some extent.
Intracerebrally administered PAH and PCG were eliminated in a saturable
manner with rate constants of 0.039 and 0.043
min–1, respectively (Figs.
3 and
4). The efflux rate of PAH
estimated in the present study was close to the reported value of 0.059
min–1 (Kakee
et al., 1997). The Km values of the
elimination of PAH and PCG from the cerebrum across the BBB were 168 and 29
µM, respectively. The Km value of the efflux of PAH was
similar to the reported value of 267 µM, taking the dilution factor of 45
in the cerebrum into account (Kakee et
al., 1997). The efflux of PCG and PAH was inhibited in a
dose-dependent manner by PAH and PCG, with Ki values of
106 and 58 µM, respectively. These values are comparable with their
Km values, indicating that PAH and PCG share the same
efflux transport system at the BBB. Furthermore, this was supported by the
inhibition study using cimetidine and pravastatin as inhibitors
(Fig. 5). The degree of
inhibition of the elimination from the cerebrum by cimetidine and pravastatin
was similar for PAH and PCG. Considering that PAH and PCG are substrates of
rOat3 and that rOat3 is expressed on the abluminal membrane of the BCEC, the
Km and Ki values determined in this
study may reflect the affinity of PAH and PCG for rOat3. Indeed, these values
were comparable with the Ki and Km
values of PAH and PCG (398 µM for PAH and 82.6 µM for PCG) for the rOat3
expression system reported previously
(Nagata et al., 2002). The
inhibitory effect of cimetidine and pravastatin, substrates of Oat3, on the
elimination of PAH and PCG from the brain across the BBB further supports the
participation of rOat3 in this process. The Ki and
Km values of cimetidine and pravastatin for rOat3 are 47
and 13 µM, respectively (Hasegawa et
al., 2002; Nagata et al.,
2002), suggesting that the degree of inhibition by these
inhibitors observed here may be reasonable, although further studies are
required to determine their in vivo Km and
Ki values. These results support our speculation that
rOat3 is involved in the elimination of PAH and PCG from the brain across the
BBB.
The apparent elimination rate constants of PAH and PCG were similar
(Fig. 3), although the
intrinsic transport activity of PCG by rOat3 was much greater than that of PAH
(Kusuhara et al., 1999;
Nagata et al., 2002). There
are two possibilities to account for this discrepancy. The first is a
difference in the distribution volume in the brain. The elimination rate
constant is obtained from the efflux clearance, an intrinsic parameter for the
efflux transport activity across the BBB, divided by the distribution volume
in the brain (Kakee et al.,
1996). If the distribution volume of PCG is much greater than that
of PAH, it is possible that the elimination rate constants of PAH and PCG are
comparable even though the efflux transport activity of PCG is greater than
that of PAH. The second is a difference in the rate of luminal secretion. The
elimination of compounds from the cerebrum into the systemic circulation
consists of two steps, i.e., uptake across the abluminal membrane and
subsequent excretion across the luminal membrane. The apparent elimination
rate constant determined by the BEI method represents a combination of these
two processes. Assuming that the rate-limiting step of the efflux of PCG from
the brain across the BBB is luminal excretion and the rate is smaller than
that of PAH, similar apparent elimination rate constants for PAH and PCG will
be observed.
A carrier-mediated influx of PCG from the blood to the brain via the BBB
has been shown by an in situ brain perfusion technique
(Suzuki et al., 1989). The
major transport system of PCG from the blood to the brain via the BBB is by a
saturable process, with a Km value of approximately 8 to
30 µM. Weak immunofluorescence signals for rOat3 were detected along the
luminal membrane of the brain capillaries
(Fig. 2). In addition, the
Km value of the brain uptake of PCG was comparable with
that of the efflux of PCG from the cerebrum across the BBB, i.e., 29 µM,
determined in this study and that for the rOat3 expression system, i.e., 82.6
µM, reported previously (Nagata et al.,
2002). Thus, it is possible that rOat3 can account for the uptake
of PCG from the blood to the brain via the BBB. This possibility should be
examined using inhibitors of rOat3 and/or Oat3 knockout mice
(Sweet et al., 2002b) to
confirm the functional involvement of rOat3 in the uptake of its substrates
from the circulating blood. Furthermore, it has been reported that rOat3
functions as an organic anion/dicarboxylate exchanger like rOat1
(Sweet et al., 2002a). It is
speculated that rOat3 can mediate both uptake and efflux across the plasma
membrane depending on the concentration gradient of the substrate and its
driving force and that rOat3 is involved in the excretion across the luminal
membrane of the brain capillaries. Another candidate among the efflux
transporters for organic anions on the luminal membrane may be multidrug
resistance associated protein 2 (Mrp2) since the expression of this
transporter on the luminal membrane of the BCEC has been reported
(Miller et al., 2000) and
ATP-dependent accumulation of PAH into membrane vesicles expressing Mrp2 has
also been reported (Van Aubel et al.,
2000). Mrp1 as well as Mrp2 would explain the efflux process since
this ABC transporter accepts PAH as a substrate
(Leier et al., 2000), although
the presence of Mrp1 at the BBB is debatable (Kusuhara and Sugiyama,
2001a,b).
Further studies are necessary to evaluate the contribution of the transporters
to the total excretion across the luminal membrane of the brain
capillaries.
A variety of compounds have been shown to be eliminated across the BBB in a
carrier-mediated manner using the BEI method. These compounds include
E217G, E1S, and indoxyl sulfate (IS)
(Hosoya et al., 2000;
Sugiyama et al., 2001;
Ohtsuki et al., 2002). PAH
inhibited the elimination of E217G and IS at a concentration
of 300 and 10 mM in the injectate, respectively. However, 100 mM PAH in the
injectate did not affect the elimination of E1S, although this
concentration is sufficient to saturate the efflux of PAH itself from the
cerebrum. Since the degree of inhibition by PAH may reflect the contribution
of rOat3, rOat3 appears to participate in the efflux of E217G
and IS, although the contribution of rOat3 to the total efflux of
E217G from the brain is approximately 20%. In contrast,
although E1S is reported to be transported by rOat3
(Kusuhara et al., 1999), PAH
had no effect on the efflux of this compound in the in vivo situation. This
discrepancy may be accounted for by the minor contribution of rOat3 to the
efflux of E1S from the brain across the BBB.
The physiological role of Oat3 has been discussed
(Kusuhara et al., 1999;
Ohtsuki et al., 2002). Since
the BCEC are connected to each other by highly developed tight junctions, it
is unlikely that hydrophilic compounds such as metabolites of
neurotransmitters are transported through the BBB via paracellular passive
diffusion. Thus, it seems that there are efflux transporters at the BBB able
to pump some hydrophilic substrates out of the brain. It has been shown that
metabolites of neurotransmitters such as dopamine, norepinephrine,
epinephrine, serotonin, and histamine are inhibitors for rOat3, suggesting
that these compounds are endogenous substrates of Oat3. rOat3 may be involved
in the elimination of endogenous waste material as well as exogenous
hydrophilic organic anions from the brain to maintain brain homeostasis. The
expression of rOat3 in the BCEC and choroid plexus, the interface between the
brain interstitial fluid or cerebrospinal fluid and the blood circulation,
supports this hypothesis. Further studies, including demonstration of the
dose-dependent elimination of hydrophilic metabolites from the brain into the
circulation across the BBB and blood-cerebrospinal fluid barrier, will be
required to identify the precise physiological function of Oat3 in the
brain.
The human homolog of rOat3, hOAT3, was isolated from a human kidney cDNA
library (Cha et al., 2001). The
substrate specificity of hOAT3 is similar to that of rOat3, and Northern blot
analysis revealed that hOAT3 mRNA is expressed in the kidney and, to the
lesser extent, in the brain and skeletal muscle. It is possible that hOAT3 is
localized at the BBB and blood-cerebrospinal fluid barrier in the brain and is
involved in regulating the concentrations of endogenous and exogenous
substrates in the central nervous system, considering the characteristics of
rOat3 in the brain reported by us and other groups
(Ohtsuki et al., 2002).
In conclusion, rOat3 is the dominant isoform of the Oat family expressed in the BCEC and plays an important role in the elimination of PAH and PCG from the cerebrum into the systemic circulation across the BBB.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: BBB, blood-brain barrier; BCEC, brain capillary
endothelial cells; BEI, brain efflux index; E217G,
17-estradiol-D-17-glucuronide; E1S,
estrone-3-sulfate; PAH, para-aminohippuric acid; Oatp, organic anion
transporting polypeptide; RT-PCR, reverse transcription-polymerase chain
reaction; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline
containing 0.05% Tween 20; BSA, bovine serum albumin; GFAP, glial fibrillary
acidic protein; ECF, extracellular fluid; PCG, benzylpenicillin; bp, base
pair; P-gp, P-glycoprotein; Mrp2, multidrug resistance associated protein 2;
IS, indoxyl sulfate.
Address correspondence to: Yuichi Sugiyama, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.utokyo.ac.jp
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