a clearance system for prostaglandin d a sleep-promoting...

JPET#197012

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A clearance system for prostaglandin D2, a sleep-promoting factor, in

the cerebrospinal fluid: role of the blood-cerebrospinal barrier

transporters

Masanori Tachikawa, Kazuhiro Tsuji, Reiji Yokoyama, Takanori Higuchi, Go

Ozeki, Ayane Yashiki, Shin-ichi Akanuma, Kazuyuki Hayashi, Akio Nishiura and

Ken-ichi Hosoya

Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical

Sciences, University of Toyama, Toyama, Japan (M.T., K.T., R.Y., T.H., G.O., A.Y., S.A.,

K.Ho.)

Division of Membrane Transport and Drug Targeting, Graduate School of

Pharmaceutical Sciences, Tohoku University, Sendai, Japan (M.T.)

Ono Pharmaceutical Co., Ltd, Osaka, Japan (K.Ha., A.N.)

JPET Fast Forward. Published on August 29, 2012 as DOI:10.1124/jpet.112.197012

Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 29, 2012 as DOI: 10.1124/jpet.112.197012

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Running title: Prostaglandin D2 clearance from the CSF

Corresponding author: Professor Ken-ichi Hosoya, Ph.D.

Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical

Sciences, University of Toyama, Toyama 930-0194, Japan. Voice: +81-76-434-7505;

FAX: +81-76-434-5172; E-mail: [email protected]

Number of text pages: 33

Number of tables: 2

Number of figures: 6

Number of references: 39

Number of words in Abstract: 199 words

Number of words in Introduction: 547 words

Number of words in Discussion: 1,358 words

Number of supplemental data: None

Abbreviations: ABCC4, ATP-binding cassette transporter C4; BBB, blood-brain

barrier; BCSFB, blood-cerebrospinal fluid barrier; CSF, cerebrospinal fluid; DHEAS,

dehydroepiandrosterone-3-sulfate; DP1, D-type prostanoid receptor 1; ECF,

extracellular fluid; MDCK, Madin-Darby canine kidney; MRP, multidrug

resistance-associated protein; OAT, organic anion transporter; oatp, organic anion

transporting polypeptide; PCG, benzylpenicillin; PG, prostaglandin; PGT, prostaglandin

transporter; SLC, solute carrier; SOS, standard oocyte saline

Recommended section: Metabolism, Transport, and Pharmacogenomics


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Abstract

Although the level of prostaglandin D2 (PGD2) in cerebrospinal fluid (CSF) affects

the action of D-type prostanoid receptors which promote physiological sleep, the

regulatory system of PGD2 clearance from the CSF is not fully understood. The purpose

of this study was to investigate PGD2 elimination from the CSF via the blood-CSF

barrier (BCSFB). The in vivo PGD2 elimination clearance from the CSF was 16-fold

greater than that of inulin, which is considered to reflect CSF bulk flow. This process

was inhibited by the simultaneous injection of unlabeled PGD2. The characteristics of

PGD2 uptake by isolated choroid plexus were, at least partially, consistent with those of

prostaglandin transporter (PGT) and organic anion transporter 3 (OAT3). Studies using

an oocyte expression system showed that PGT and OAT3 were able to mediate PGD2

transport with a Michaelis-Menten constant of 1.07 μM and 7.32 μM, respectively.

RT-PCR and immunohistochemical analyses revealed that PGT was localized on the

brush-border membrane of the choroid plexus epithelial cells. These findings indicate

that the system regulating the PGD2 level in the CSF involves PGT- and

OAT3-mediated PGD2 uptake by the choroid plexus epithelial cells, acting as a pathway

for PGD2 clearance from the CSF via the BCSFB.


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Introduction

Prostaglandin (PG) D2 is an endogenous sleep-promoting substance which regulates

physiological sleep, i.e., both non-rapid eye movement (NREM) and rapid eye

movement (REM) sleep, through D-type prostanoid receptor 1 (DP1 receptor) (Huang et

al., 2007). Since the DP1 receptors are predominantly localized in the arachnoid

trabecular cells in the ventral surface of the rostal basal forebrain (Mizoguchi et al.,

2001), which faces the CSF, the CSF concentration of PGD2 will be a key determinant

of the action of DP1 receptors. Indeed, infusion of PGD2 into the lateral ventricle

increases the amount of NREM sleep in a dose-dependent manner in wild-type mice but

not in DP1 receptor knockout mice (Mizoguchi et al., 2001). In unanesthetized

conscious rats, the CSF concentration of PGD2 exhibits a significant circadian

fluctuation with a peak (1197 pg/mL; 3.4 nM) at 2 pm and a minimum (438 pg/mL; 1.2

nM) at 6 am (Pandey et al., 1995). These lines of evidence imply that regulatory

systems for the generation and/or elimination of PGD2 in the CSF produce a circadian

rhythm.

The rates of generation and elimination of PGD2 in the CSF appear to be well

balanced to maintain PGD2 levels in the CSF. It has been proposed that PGD2 is

generated from PGH2 mainly by lipocalin-type prostaglandin D synthetase (L-PGDS)

and released into the CSF (Qu et al., 2006). PGD2 is also transported from the

circulating blood to the brain across the blood-brain barrier (BBB) (Suzuki et al., 1986).

However, a clearance system for PGD2 from the CSF would be essential for regulating

PGD2 levels in the CSF. In the brain, it is unlikely that prostaglandins are effectively

inactivated because there is little expression and activity of 15-hydroxyprostaglandin

dehydrogenase, the rate-limiting enzyme for prostaglandin catabolism, in the adult brain

(Alix et al., 2008). It is thus conceivable that the primary pathway for removing PGD2

from the CSF is the CSF-to-blood vectorial transport across the blood-CSF barrier


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(BCSFB) which is formed by the complex tight junctions of choroid plexus epithelial

cells in the ventricle (Hosoya et al., 2004). In support of this notion, it has been reported

that the choroid plexus takes up PGF2α via a saturable transport process (DiBenedetto

and Bito, 1986).

Cellular transport of PGs is mediated by several transporters, i.e., organic anion

transporter (OAT/SLC22) (Kimura et al., 2002), organic cation transporter

(OCT/SLC22) (Kimura et al., 2002), and organic anion transporting polypeptide

(OATP/SLCO) (Kanai et al., 1995; Cattori et al., 2001). Among high-affinity

transporters for PGs with nanomolar Km values, OAT3 (SLC22A3) (Nagata et al.,

2002) and prostaglandin transporter (PGT/SLCO2A1) (Kis et al., 2006) are expressed in

the choroid plexus. And OAT3 recognizes PGE2 with a Km value of 345 nM (Kimura et

al., 2002) while PGT transports various prostaglandins, including PGE1, PGE2, and

PGF2α, with a Km value of 70-104 nM (Kanai et al., 1995). Because there is an overlap

in substrates between PGT and OAT3, a combination of these transporters at the BCSFB

should act as a regulatory system for PGD2 levels in the CSF.

The purpose of the present study was to investigate the elimination of PGD2 from the

CSF across the BCSFB using intracerebroventricular administration and the uptake by

isolated choroid plexus, and to identify which transporters are responsible for PGD2

transport using an expression system involving of Xenopus laevis oocytes.


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Materials and methods

Animals

Adult male ddY mice (25-30 g), Wistar rats (260-280 g), and female Hartley

guinea-pigs (300-350 g) were purchased from Japan SLC (Hamamatsu, Japan). Mature

female Xenopus laevis oocytes were purchased from Kato-S-Science (Chiba, Japan).

They were maintained in a controlled environment and all experiments were approved

by the Animal Care Committee, University of Toyama. In order to minimize the diurnal

variations, the transport analyses and tissue sampling were conducted at

12:00pm-7:00pm.

Reagents

[5,6,8,9,12,14,15-3H(N)]prostaglandin D2 ([3H]PGD2, 198.8 Ci/mmol) was

obtained from Perkin-Elmer Life and Analytical Sciences (Boston, MA, USA).

[Carboxyl-14C]Inulin ([14C]inulin 1.9 mCi/g) was obtained from Moravek Biochemicals

(Brea, CA, USA). n-[1-14C]Butanol ([14C]butanol, 5.0 mCi/mmol) was obtained from

American Radiolabeled Chemicals (St. Louis, MO, USA). All other chemicals were

commercial products of analytical grade.

In vivo PGD2 efflux from the CSF after intracerebroventricular administration

[3H]PGD2 elimination from the CSF after intracerebroventricular

administration was studied using the method described previously (Tachikawa et al.,

2008a; Tachikawa et al., 2008b). Male Wistar rats were anaesthetized by intraperitoneal

injection of pentobarbital (50 mg/kg body weight) and their heads were fixed in a

stereotaxic apparatus (Narishige, Tokyo, Japan). A hole was drilled in the skull, 1.5 mm

to the left and 0.5 mm posterior to the bregma, into which a needle was fixed as a


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cannula for injection. A cerebroventricular dose of [3H]PGD2 (0.4 μCi/rat) and

[14C]inulin (0.005 μCi/rat) dissolved in 10 μL extracellular fluid (ECF) buffer

containing 122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 0.4 mM K2HPO4, 1.4 mM

CaCl2, 1.2 mM MgSO4, 10 mM D-glucose and 10 mM HEPES was administered to the

left lateral ventricle through the cannula. At designated times, CSF (50-100 μL) was

withdrawn by cisternal puncture. The remaining radioactivity in the CSF specimens was

determined using a liquid scintillation spectrophotometer (LSC-5000, Aloka, Tokyo,

Japan). The kinetic parameters for the elimination of [3H]PGD2 and [14C]inulin were

determined from eqn. 1 using the non-linear least-square regression analysis program,

MULTI (Yamaoka et al., 1986):

CCSF (t) = dose / Vd x exp (-kel x t) (Eq. 1)

where CCSF (t), Vd and kel are the CSF concentration at time t, the volume of CSF and

the elimination rate constant, respectively. As indicated in Fig. 1a, the y-intercept was

expressed as % dose per mL CSF. Since the y-intercept value of [3H]PGD2 and

[14C]inulin in Fig. 1a was ~500 % dose/mL CSF, the volume of CSF in which

[14C]inulin can be initially distributed was determined to be approximately 200 μL. This

volume is in close agreement with the total CSF volume in rats as reported previously

(Cserr and Berman, 1978). An apparent elimination clearance was determined by

multiplying kel by Vd (kel x Vd).

HPLC Analysis

At 2 min after intracerebroventricular administration of [3H]PGD2 (2 μCi/rat), the

CSF was collected via cisternal puncture. The CSF was mixed with acetonitrile and

acetic acid each at a concentration of 45% (v/v) and 0.1% (v/v), respectively. The

mixture was centrifuged at 12,000g for 5 min at 4°C, and the supernatant was collected.

Then, 20 μl was subjected to HPLC analysis. The HPLC system consisted of a pump


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(EP-300; Eicom, Kyoto, Japan), a column oven (ATC-300; Eicom). The HPLC

analytical column was an Inertsil ODS-2 column (5 μm, 4.6 x 150 mm; GL Sciences,

Tokyo, Japan). The mobile phase (acetonitrile-acetic acid-water, 45:0.1:55, v/v/v) was

pumped through the column at a rate of 1.0 ml/min at 20°C. Samples of eluate were

collected in scintillation counting vials, and the 3H radioactivity in each fraction (1.0

ml) was determined in a liquid scintillation counter (LSC-5000; Aloka).

PGD2 uptake using freshly isolated rat choroid plexus

The uptake of [3H]PGD2 by rat choroid plexus was examined using the

centrifugal filtration method described previously (Tachikawa et al., 2008a; Tachikawa

et al., 2008b). The rats were decapitated and the choroid plexus was isolated from the

lateral ventricles and incubated at 37°C for 1 min in 300 μL ECF buffer. Incubation

medium containing both [3H]PGD2 and [14C]butanol in the presence or absence of

inhibitors was added to initiate uptake. The final concentration of incubation medium

was: [3H]PGD2 (30 nM) and [14C]butanol (300 μM). The radioactivity in the specimens

was determined using a liquid scintillation spectrophotometer (LSC-5000, Aloka). The

distribution volume of [3H]PGD2 per μL choroid plexus (μL/μL choroid plexus) was

calculated as the amount of [3H]PGD2 accumulated per μL isolated choroid plexus

(dpm/μL choroid plexus) divided by the medium concentration of [3H]PGD2 (dpm/μL).

The volume (μL) of the isolated choroid plexus was calculated as the amount of

[14C]butanol accumulated per the isolated choroid plexus (dpm/the isolated choroid

plexus) divided by the medium concentration of [14C]butanol (dpm/μL).

Uptake using PGT- and OAT3-expressing Xenopus laevis oocytes

Using T7 RNA polymerase, capped cRNA was transcribed from NotI-linearized

pGEM-HEN containing an open reading frame of mouse PGT and rat OAT3 cDNAs as


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described elsewhere (Mori et al., 2003). Defolliculated oocytes were injected with 23 nL

water or the capped cRNA (50-100 ng) and incubated at 18°C in freshly prepared

standard oocyte saline (SOS) solution containing 100 mM NaCl, 2 mM KCl, 1.8 mM

CaCl2, 1 mM MgCl2, and 5 mM HEPES, 25 μg/mL gentamycin, 2.5 mM pyruvic acid

and 1% bovine serum albumin, pH 7.5. The SOS solution used to incubate the oocytes

was replaced daily with fresh solution. Experiments were performed after incubation for

4 to 6 days. An uptake study using PGT- and OAT3-expressing Xenopus laevis oocytes

was performed as described previously (Mori et al., 2003). The oocytes injected with

PGT cRNA or OAT3 cRNA were preincubated with 500 μL ND96 solution containing

96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4, for

20 min at 20°C before the uptake experiment. The uptake experiment was initiated by

replacing the ND96 solution with 200 μL of the same solution containing 1.2 nM

[3H]PGD2 and terminated by addition of ice-cold ND96 solution after incubation for a

designated time at 20°C. Oocytes were then washed four times with ice-cold ND96

solution and solubilized in 5% sodium dodecyl sulfate solution, and the accumulated

radioactivity was determined in a liquid scintillation counter (LSC-5000, Aloka). For

the inhibition study, the uptake of 1.25 nM [3H]PGD2 was measured in the presence or

absence of various unlabeled compounds in ND96 solution. The distribution volume of

[3H]PGD2 per oocyte (μL/oocyte) was calculated as the amount of [3H]PGD2

accumulated per oocyte (dpm/oocyte) divided by the medium concentration of

[3H]PGD2 (dpm/μL). The specific uptake was calculated by subtracting the uptake by

water-injected oocytes from the uptake by PGT- or OAT3-expressing oocytes.

Kinetic analyses

The kinetic parameters for PGD2 uptake by Xenopus oocytes expressing PGT and

OAT3 were obtained from the following eqn 2:


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V = (Vmax x S) / (Km + S) (Eqn 2)

where V is the uptake rate of PGD2, S is the PGD2 concentration in the medium, Km is

the Michaelis-Menten constant, and Vmax is the maximum uptake rate. To obtain

kinetic parameters, the equation was fitted using the iterative non-linear least-squares

regression analysis program, MULTI (Yamaoka et al., 1986).

RT-PCR analysis

Total RNA was isolated from mouse choroid plexus and rat brain and lung

using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer.

The RNA was reverse-transcribed using oligo(dT) primer and ReverTra Ace (Toyobo,

Osaka, Japan). PCR was performed using ExTaq DNA polymerase (Takara, Kyoto,

Japan) with the following thermal cycle program: 30 cycles of 94°C for 30 s, 60°C for

30 s, 72°C for 1 min, and a final period of 72°C for 10 min. The sequences of the

specific primers for mouse PGT (GenBank Accession number: AF323958) were as

follows: the sense sequence was 5’-CATCTTCGTCAGCTACTTCGGC-3’ and the

antisense sequence was 5’-CACATGCTGCTGGTCTCCTTCT-3’. The PCR products

were separated by electrophoresis on an agarose gel in the presence of ethidium

bromide and visualized under ultraviolet light. The molecular identify of the resultant

product was confirmed by sequence analysis using a DNA sequencer (ABI PRISM

3100; PE-Applied Biosystems, Foster City, CA, USA).

Antibodies

Polyclonal antibodies to PGT and L-PGDS were raised against amino acid residues

122-161 of mouse PGT (GenBank accession number: AF323958) and 150-189 of

mouse L-PGDS (GenBank accession number: NP032989). The polypeptides were

expressed as glutathione S-transferase (GST) fusion proteins using the pGEX4T-2


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plasmid vector (GE Healthcare, Chalfont St. Giles, UK). The fusion protein was

purified with glutathione-Sepharose 4B (GE Healthcare), emulsified with Freund’s

complete adjuvant (Difco, Detroit, MI, USA), and injected subcutaneously into female

Hartley guinea-pigs at intervals of 2 weeks. Two weeks after the sixth injection,

affinity-purified antibodies were prepared, first using protein G-Sepharose (GE

Healthcare) and then using antigen peptides coupled to cyanogen bromide-activated

Sepharose 4B (GE Healthcare). For the preparation of affinity media, polypeptides free

of GST were obtained by elution of the cleaved polypeptide after in-column thrombin

digestion of fusion proteins bound to glutathione-Sepharose 4B.

Immunoblotting

Mouse lung and choroid plexus were homogenized in a buffer containing (in mM):

10 HEPES, 1 EDTA, 1 EGTA, 320 sucrose, 1 phenylmethylsulfonyl fluoride, and a

protease inhibitor cocktail (Sigma Aldrich, St Louis, MO, USA), pH 7.4. Mouse brain

was homogenized in an RIPA buffer containing: 10 mM Tris-HCl, pH 7.4, 1 mM EDTA,

150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, and a protease

inhibitor cocktail (Sigma Aldrich). The homogenates were centrifuged at 10,000 g for

15 min. The supernatants were then obtained as whole lysate protein samples or further

centrifuged at 100,000 g for 60 min to obtain a crude membrane fraction from the

pellets. The protein concentration was determined by the Bradford method using a DC

Protein Assay kit (Bio-rad, Hercules, CA, USA). Protein samples (50 μg per lane) were

fractionated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

and electroblotted onto a nitrocellulose membrane. The blotted membrane was

incubated with an affinity-purified PGT antibody at 0.1 μg/mL or affinity-purified

L-PGDS antibody at 0.5 μg/mL in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 8.0

and 125 mM NaCl, pH 7.4) containing 0.1% Tween 20 and 4% skimmed milk, and


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visualized with an enhanced chemiluminescence kit (GE Healthcare).

Immunohistochemistry

Preparation of brain paraffin sections (5 μm) using a sliding microtome (SM2000R;

Leica, Nussloch, Germany) and immunohistochemistry were performed as described

previously (Tachikawa et al., 2004). In brief, for immunofluorescence, sections were

immunoreacted overnight with guinea-pig antibody to PGT or L-PGDS (2 μg/mL)

singly or in combination with rabbit glucose transporter 1 (GLUT1) antibody (0.5

μg/mL, (Sakai et al., 2003)) and mouse Na+, K+-ATPase antibody (2 μg/mL, Millipore,

Billerica, CA, USA). Subsequently, they were incubated with species-specific Alexa

Fluor 488- (Invitrogen) and Cy3-conjugated secondary antibodies (Jackson

ImmunoResearch, West Grove, PA, USA) for 2 h. Photographs were taken using a

confocal laser scanning microscope (TCS-SP5; Leica).

Statistical analysis

Unless otherwise indicated, all data are presented as the mean±SEM. The kinetic

parameters are presented as the mean±SD. An unpaired, two-tailed Student’s t-test was

used to determine the significance of differences between two group means. One-way

analysis of variance followed by the modified Fisher’s least-squares difference method

was used to assess the statistical significance of differences among means of more than

two groups.


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Results

Elimination of PGD2 from rat CSF after intracerebroventricular administration

Figure 1a shows the residual CSF concentration of [3H]PGD2 and [14C]inulin after

intracerebroventricular administration as a function of time. [3H]PGD2 was eliminated

from the CSF with a higher rate constant of 0.604±0.118 min-1 than that of [14C]inulin

(0.0397±0.0241 min-1), a reference compound for CSF turnover and diffusion into the

brain interstitional space through the ependymal space. The half-life of [3H]PGD2 and

[14C]inulin elimination from the CSF was estimated to be 1.15 min and 17.5 min,

respectively. The elimination clearance of PGD2 from the CSF (124 μL/min) was

approximately 16-fold higher than that of inulin (7.80 μL/min). The elimination

clearance of inulin was close to the CSF bulk flow rate (2.9 μL/min) obtained by Suzuki

et al. (1985). The simultaneous injection of unlabeled PGD2 with [3H]PGD2 at a CSF

concentration of 100 μM resulted in a 3.4-fold increase in the residual concentration of

[3H]PGD2 (340% dose/mL CSF) compared with that in the absence of PGD2 (101%

dose/mL CSF) at 20 min.

Figure 1b and 1c shows representative HPLC chromatograms of 3H radioactivity in

the injectate of [3H]PGD2 and the CSF at 2 min after intracerebroventricular

administration of [3H]PGD2, respectively. One major peak which corresponds to intact

[3H]PGD2 was detected at the elution time of 7.5 min in the injectate. At 2 min after

intracerebroventricular administration of [3H]PGD2, two major peaks were detected in

the CSF at the same elution time as intact [3H]PGD2 and the earlier elution time (3.5

min). According to the paper by Eguchi et al. (1992), the additional peak was

presumably considered to correspond to PGF2α. Since the sum of radioactivity eluted

from 6.5 to 9.5 min, which corresponds to intact [3H]PGD2, showed 73.2% of the total

radioactivity, most of [3H]PGD2 administrated in the CSF would undergo the


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elimination as the intact form.

Characteristics of PGD2 uptake by isolated rat choroid plexus

To examine whether PGD2 was eliminated from the CSF via the BCSFB, a study of

[3H]PGD2 uptake by the isolated rat choroid plexus was performed. [3H]PGD2 uptake

by the isolated choroids plexus exhibited a time-dependent increase linearly for up to 1

min of incubation with an initial uptake rate of 4.47±0.56 μL/(min•μL choloid plexus)

(Fig. 1d). To characterize the transporter(s) involved in [3H]PGD2 uptake by the isolated

choroid plexus, the inhibitory effects of various compounds on the [3H]PGD2 uptake

were investigated (Table 1). PGD2 and PGB1 produced 78% and 85% inhibition of the

[3H]PGD2 uptake at a concentration of 1 mM. Bromocresolgreen, an inhibitor of PGT,

inhibited the uptake by 79% at a concentration of 1 mM. The uptake was also inhibited

by OAT3 substrates and/or inhibitors, such as dehydroepiandrosterone-3-sulfate

(DHEAS), indomethacin, benzylpenicillin, and probenecid, by 23-75%. In contrast,

L-lactic acid had little effect on the uptake. These results suggest the involvement of

PGT and OAT3 in the PGD2 uptake by the isolated rat choroid plexus.

Characteristics of PGT and OAT3-mediated [3H]PGD2 uptake in mouse PGT and

rat OAT3-expressing Xenopus oocytes

Although the [3H]PGD2 uptake study using the isolated choroid plexus suggests an

involvement of PGT and OAT3, the contribution of each transporter to the PGD2 uptake

remains unknown. In order to determine the characteristics of PGT- and OAT3-mediated

PGD2 transport, [3H]PGD2 uptake was studied with PGT- and OAT3-expressing oocytes

(PGT/oocytes and OAT3/oocytes, respectively). [3H]PGD2 was taken up in a

time-dependent manner by PGT/oocytes (Fig. 2a) and OAT3/oocytes (Fig. 3a), and the

uptake by these oocytes was several-fold higher than in water-injected oocytes. As


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shown in Figure 2b and 3b, PGD2 uptake by PGT/oocytes and OAT3/oocytes exhibited

saturable kinetics with a Km of 1.07±0.32 μM and a Vmax of 9.36±0.71

pmol/(h•oocyte), and a Km of 7.32±1.27 μM and a Vmax of 2.14±0.28 pmol/(h•oocyte),

respectively. The inhibitory effects of various compounds on PGT- and OAT3-mediated

[3H]PGD2 uptake were examined (Table 2). Bromocresolgreen, at a concentration of 1

mM, produced a marked inhibition of the PGT-mediated [3H]PGD2 uptake whereas it

had the lesser effect on the OAT3-mediated uptake. In contrast, benzylpenicillin, at a

concentration of 1 mM, significantly inhibited the OAT3-mediated [3H]PGD2 uptake

whereas it had no effect on the PGT-mediated [3H]PGD2 uptake. DHEAS, at a

concentration of 1 mM, had a similar inhibitory effect on the PGT- and OAT3-mediated

[3H]PGD2 uptake. OAT3 substrates and/or inhibitors, such as probenecid, and

estradiol-17β-glucuronide, inhibited the PGT-mediated [3H]PGD2 uptake by 17-21%.

Indomethacin, p-aminohippuric acid, and diclofenac inhibited OAT3-mediated

[3H]PGD2 uptake by 18-61%. L-Lactic acid had a little effect on PGT-mediated

[3H]PGD2 uptake.

Expression and localization of PGT in the choroid plexus

RT-PCR gave one amplified product at the expected size of 255 bp in the mouse

choroid plexus as well as the brain and lung used as a positive control for PGT

expression (Fig. 4). The nucleotide sequences of the products were identical with that of

mouse PGT (GenBank accession number: AB103401). The expression and localization

of PGT protein in the choroid plexus were characterized with guinea-pig PGT antibody.

Immunoblot analysis showed a single band at ~55-65 kDa which was detected in the

lung used as a positive control for PGT expression, and the choroid plexus (Fig. 5a and

5b). One possible explanation for the difference in the molecular size of PGT between

the lung and choroid plexus is the glycosylation between the lung and choroid plexus


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PGT. By using immunohistochemical techniques, the PGT antibody labeled the choroid

plexus (Fig. 6a): PGT immunofluorescence was intense on the CSF side membrane of

the choroid plexus epithelial cells (Fig. 6c). The bands and immunochemical staining

were abolished with the use of antibody with preabsorbed antigen peptides (100 μg/mL),

indicating the specificity of PGT antibody (Fig. 5a and 6d). By double

immunofluorescence for Na+, K+-ATPase, a brush-border membrane marker of choroid

plexus epithelial cells, the PGT immunoreactivities showed extensive overlapping with

those of Na+, K+-ATPase (Fig. 6e). In contrast, they were negative for GLUT1, a

basolateral membrane marker of choroid plexus epithelial cells (Fig. 6f). These results

indicate that PGT is localized on the brush-border membrane of choroid plexus

epithelial cells. Guinea-pig L-PGDS antibody recognized a single band at 26 kDa (Fig.

5c) which is consistent with previous results (Urade et al., 1989). The band was

abolished with the use of antibody with preabsorbed antigen peptides (100 μg/mL),

indicating the specificity of L-PGDS antibody (Fig. 5c). The L-PGDS antibody strongly

labeled the leptomeninges (Fig. 6b).


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Discussion

In the present study, our findings indicate that a regulatory system of the PGD2 level

in the CSF involves PGT- and OAT3-mediated PGD2 uptake by the choroid plexus

epithelial cells, acting as a pathway for PGD2 elimination from the CSF.

[3H]PGD2, after intracerebroventricular administration, was removed from the CSF

most likely as the intact form with a 16-fold greater elimination clearance than that of

[14C]inulin, which is considered to reflect the CSF bulk flow (Fig. 1a). The rapid

elimination of [3H]PGD2 from the CSF was inhibited by simultaneous injection of

unlabeled PGD2 at a CSF concentration of 100 μM (Fig. 1a), suggesting active efflux

transport of PGD2 from the CSF. This is in close agreement with a previous report

demonstrating active elimination of PGF2α from rabbit CSF using ventriculo-cisternal

perfusions (Bito et al., 1976). To evaluate the role of the BCSFB in the PGD2

elimination from CSF, [3H]PGD2 uptake by the freshly isolated rat choroid plexus was

investigated (Fig. 1d and Table 1). [3H]PGD2 undergoes concentrative uptake by the

choroid plexus and this is inhibited by unlabeled PGD2 (Fig. 1d). This indicates that the

BCSFB contributes to the carrier-mediated efflux transport of PGD2 from the CSF. The

elimination clearance of PGD2 from the CSF via the BCSFB was estimated from the

initial uptake rate of PGD2 by the isolated choroid plexus and found to be 26.8 μL/min

per rat [4.47 μL/(min•μL choroid plexus) x 6 μL (the volume of total rat choroid plexus

per rat; Ogawa et al., 1994)]. The PGD2 efflux transport via the BCSFB makes a 21.6%

contribution to the total PGD2 elimination clearance from the CSF in vivo (124 μL/min

per rat). Since the elimination clearance of inulin from the CSF was 7.80 μL/min per rat,

i.e., 6.29% of the total PGD2 elimination clearance from the CSF, the contribution of the

CSF bulk flow and diffusion into the brain interstitial space appears to be limited. It has

been reported that Slco2b1 (moat1) transports PGD2 and is expressed in the brain

parenchymal cells (Nishio et al., 2000). It would raise another possibility that


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Slco2b1-mediated uptake by brain parenchymal cells as a potential elimination pathway

of PGD2 in the CSF.

The effects of several inhibitors on [3H]PGD2 uptake by the isolated choroid plexus

were examined to identify the transporters involved (Table 1). Uptake studies with

PGT- and OAT3-expressing Xenopus oocytes showed that bromocresolgreen and

DHEAS inhibit both PGT- and OAT3-mediated [3H]PGD2 uptake whereas

benzylpenicillin only inhibits OAT3-mediated [3H]PGD2 uptake (Table 2). The

inhibition ratio of bromocresolgreen and DHEAS for the [3H]PGD2 uptake (75-80%) by

the choroid plexus is greater than that of benzylpenicillin (54%), each at a concentration

of 1 mM (Table 1). Furthermore, although the Ki value of probenecid for OAT3 was

reported to be 4.43 μM-20 μM (Sugiyama et al., 2001; Nagata et al., 2002), probenecid

inhibited the [3H]PGD2 uptake by the choroid plexus only by 23% at a concentration of

100 μM (Table 1). Because probenecid at a concentration of 1 mM had little inhibitory

effect on the PGT-mediated [3H]PGD2 uptake (Table 2), it is likely that both PGT and

OAT3 are mediators of PGD2 uptake by the choroid plexus. In support of this notion, the

present study indicates that PGT is localized on the brush-border membrane of choroid

plexus epithelial cells (Fig. 6). It should also be noted that DHEAS, PGE2 and

indomethacin are a transportable substrate (Cattori et al., 2001) and/or an inhibitor

(Kusuhara et al., 2003), respectively, of oatp1a5 (Slco1a5) which is localized on the

brush-border membrane of choroid plexus epithelial cells (Ohtsuki et al., 2003; Ohtsuki

et al., 2004). It is thus intriguing in the future study to pursue the contribution of OAT3,

PGT, and oatp1a5.

The Km values of PGD2 transport mediated by PGT (1.07 μM; Fig. 2b) and OAT3

(7.32 μM; Fig. 3b) are almost 3 orders of magnitude greater than the CSF concentration

of PGD2 (1-3 nM; (Pandey et al., 1995), PGE2 (1.2 nM; (Gao et al., 2009). This

suggests that PGT and OAT3 mediate the PGD2 uptake by the choroid plexus epithelial


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cells without saturation, thus producing continuous removal of PGD2 from the CSF.

Since the circadian fluctuation of the PGD2 concentration in the CSF ranges from 1-3

nM (Pandey et al., 1995), this may not be due to the changes in the function of PGT-

and OAT3-mediated PGD2 elimination from the CSF at the BCSFB. It has been reported

that the inhibition of PGD2-generating enzyme L-PGDS results in a reduction in the

brain PGD2 content and a suppression of sleep in rats, indicating the crucial role of this

enzyme in the regulation of physiological sleep (Qu et al., 2006). Immunohistochemical

analysis reveals that L-PGDS is highly expressed in the leptomeninges facing the CSF

(Fig. 6), which is consistent with previous reports (Urade et al., 1993). Taken these

findings into consideration, it seems likely that the circadian fluctuation in the PGD2

level in the CSF depends on the local production of PGD2, presumably via L-PGDS.

There are two types of CSF-to-blood efflux transport at the BCSFB: (i) influx transport

across the brush-border (apical) membrane of choroid plexus epithelial cells from the

CSF into the cells and (ii) subsequent efflux transport across the basolateral membrane

from the cells into the blood. The present findings suggest that PGT and OAT3 on the

brush-border membrane are involved in the PGD2 uptake from the CSF into choroid

plexus epithelial cells. It has been reported that polarized apical localization of PGT in

Madin-Darby canine kidney (MDCK) cells induces a 100-fold increase in the

apical-to-basal PGE2 transport (Endo et al., 2002). Chan et al. (2002) have reported that

PGT-mediated uptake of PGs is coupled to the efflux of intracellular lactate in

PGT-overexpressing HeLa cells. This raise a possibility that the PGT-mediated

concentrative PGD2 transport in the CSF-to-choroid plexus epithelial cells direction

would be produced by the outward lactate gradient, although the lactate gradient in the

epithelial cells remains to be determined. Furthermore, the polarized choroid plexus

epithelial cells produce transcellular transport of PGE2 in the apical-to-basolateral

direction without the inactivation of PGE2 in the cells (Khuth et al., 2005). In this regard,


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PGT and OAT3 on the brush-border membrane of choroid plexus epithelial cells would

be a key determinant of the CSF-to-blood PGD2 efflux transport across the BCSFB. On

the other hand, multidrug resistance-associated protein 4/ATP-binding cassette

transporter C4 (MRP4/ABCC4) is localized on the basolateral membrane of choroid

plexus epithelial cells (Leggas et al., 2004) and mediates active efflux transport of

prostaglandins, such as PGE1 and PGE2, out of the cells (Reid et al., 2003). Thus, MRP4

would be a potent candidate transporter for mediating PGD2 efflux transport from the

choroid plexus epithelial cells into the blood although further studies are needed to

clarify whether PGD2 is a substrate of MRP4.

The continuous inhibition of the clearance of PGD2 at the BCSFB may modify the

PGD2 level in the CSF, thus affecting physiological sleep. Kis et al. (2006) have

reported that lipopolysaccharide stimulation diminishes the luminal membrane

localization of PGT in the cerebral endothelial cells. If this were true for PGT

localization in the choroid plexus epithelial cells, PGT-mediated elimination of PGD2

from the CSF might be reduced under inflammatory conditions. Some commonly used

non-steroidal anti-inflammatory drugs, such as indomethacin and ibuprofen, have been

shown to inhibit the transport of PGF2α in the choroid plexus (Bito and Salvador, 1976).

The present study indicates that indomethacin and diclofenac inhibit OAT3-mediated

PGD2 transport (Table 2). Furthermore, we have found that cefmetazole, cefazolin, and

ceftriaxone, and cefotaxime, administered intravenously to mice, inhibit [3H]PGE2

efflux transport from the brain across the BBB most likely due to the inhibition of

MRP4 (Akanuma et al., 2010). Thus, the inhibitory effect on the PGT- and

OAT3-mediated PGD2 transport should be taken into consideration in the development

of new drugs.

In conclusion, PGT and OAT3 at the BCSFB represent the primary mechanisms for

the continuous removal of locally produced PGD2 from the CSF, thereby regulating


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PGD2 levels in the CSF. The present findings thus provide a novel insight into the

mechanisms of PGs clearance from the CSF and may be helpful in the development of

new therapeutic targets for insomnia.


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Acknowledgement

We would like to thank Drs T. Abe and T. Terasaki for supplying the

pGEM-HEN vector for protein expression in Xenopus laevis oocytes.


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Authorship Contributions

Participated in research design: Tachikawa, Tsuji, Akanuma, Hayashi, Nishiura, and Hosoya

Conducted experiments: Tachikawa, Tsuji, Yokoyama, Higuchi, Ozeki, Yashiki, and Akanuma

Contributed new reagents or analytic tools: Hayashi and Nishiura

Performed data analysis: Tachikawa, Tsuji, Yokoyama, Higuchi, Ozeki, Yashiki, and Akanuma

Wrote or contributed to the writing of the manuscript: Tachikawa, Akanuma, and Hosoya


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Footnotes

This study was supported, in part, by a Grant-in-Aid for Scientific Research from the

Japan Society for the Promotion of Science (JSPS).


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Figure legends

Figure 1 In vivo elimination of PGD2 from the CSF across the BCSFB. (a) CSF

concentration versus time profiles of [3H]PGD2 (open circle) and [14C]inulin (open

square) after intracerebroventricular administration. Simultaneous injection of unlabeled

PGE2 at a CSF concentration of 100 μM significantly inhibited the elimination of

[3H]PGE2 (closed circle). Each point represents the mean±SEM (n=3). A tracer dose of

[3H]PGD2 and [14C]inulin was administered into the lateral ventricle of rats. The

concentration of [3H]PGD2 remaining in the cisternal CSF was determined at the

designated times. [14C]Inulin was used as a reference for CSF turnover and passive

diffusion into the brain parenchyma. The values are expressed as the percentage of the

dose remaining per milliliter of CSF. *p<0.01, significantly different from control. (b

and c) Representative HPLC chromatograms of 3H-radioactivity in the injectate of

[3H]PGD2 (b) and the CSF (c) at 2 min after intracerebroventricular administration of

[3H]PGD2. Each point represents the radioactivity measured in the corresponding

fraction (1.0 ml/each). (d) Time-course of [3H]PGD2 uptake by isolated rat choroid

plexus. Choroid plexus was incubated with [3H]PGD2 (30 nM) at 37°C. Each point

represents the mean±SEM (n= 3).

Figure 2 Characteristics of PGD2 transport in PGT-expressing Xenopus oocytes

(PGT/oocytes). (a) Time-courses of [3H]PGD2 uptake (1.0 nM) by Xenopus oocytes

injected with water (closed circle), and mouse PGT cRNA (open circle). The uptake was

measured at the indicated incubation times. Each point represents the mean±SEM

(n=14-15). (b) Concentration-dependence of [3H]PGD2 uptake by PGT/oocytes. The

uptake was measured at the indicated concentrations for 1 h. The inset graph shows the

Eadie-Scatchard plot of the same data. Each point represents the mean±SEM (n=


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14-15).

Figure 3 Characteristics of PGD2 transport in OAT3-expressing Xenopus oocytes

(OAT3/oocytes). (a) Time-courses of [3H]PGD2 uptake (1.0 nM) by Xenopus oocytes

injected with water (closed circle), and rat OAT3 cRNA (open circle). The uptake was

measured at the indicated incubation times. Each point represents the mean±SEM

(n=6-10). (b) Concentration-dependence of [3H]PGD2 uptake by OAT3/oocytes. The

uptake was measured at the indicated concentrations for 1 h. The inset graph shows the

Eadie-Scatchard plot of the same data. Each point represents the mean±SEM (n=

6-10).

Figure 4 RT-PCR analysis of PGT in choroid plexus. “+” and “-” indicate RT (+) and

RT (-), respectively. The PCR products were detected at the expected size of 255 bp in

the mouse choroid plexus as well as the brain and lung used as a positive control for

PGT expression.

Figure 5 Immunoblot with guinea-pig antibodies to PGT (a and b) and L-PGDS (c).

(a) Expression of PGT protein in the lung. PGT antibody was used after being

preincubated in the absence [antigen(-)] or presence [antigen(+)] of PGT antigen for 12

h at 4°C. (b) Expression of PGT protein in the choroid plexus. (c) Expression of

L-PGDS protein in the brain. L-PGDS antibody was used after being preincubated in

the absence [antigen(-)] or presence [antigen(+)] of L-PGDS antigen for 12 h at 4°C.

The size of marker proteins is indicated to the left.

Figure 6 Immunofluorescence with guinea-pig PGT and L-PGDS antibodies. (a)

PGT immunofluorescence (green) in the choroid plexus. (b) Intense L-PGDS


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immunolabeling (red) on the arachnoid membrane. (c) Intense PGT immunoreactivities

(green) on the CSF side membrane (arrowheads) of the choroid plexus epithelial cells.

(d) Abolished PGT immunostaining in the choroid plexus with preabsorbed antibody. In

b-d, nuclei were stained by DAPI (blue, a1, a2, and b) or propidium iodide (red, c and d).

(e) PGT (red, e1 and e3) overlapped with Na+, K+-ATPase (green, e2 and e3) on the

brush-border membrane (arrowheads) of choroid plexus epithelial cells. (f) Lack of PGT

immunostaining (red, f1 and f3) in GLUT1-positive basolateral membrane (green, f2 and

f3) of choroid plexus epithelial cells. Arrowheads and arrows indicate the brush-border

membrane and the basolateral membrane, respectively. Scale bars: a, b, d; 50 μm, c, e, f;

10 μm.


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Tables

Table 1 Effect of several compounds on [3H]PGD2 uptake by isolated rat choroid

plexus

Inhibitors Concentration of

inhibitor (mM)

% of control

Control

Prostaglandin D2 (PGD2)

Prostaglandin B1 (PGB1)

Bromocresolgreen

Dehydroepiandrosterone-3-sulfate (DHEAS)

Benzylpenicillin

Indomethacin

Probenecid

L-Lactic acid

0.1

1

0.1

1

1

1

1

0.1

0.1

25

100±2

43.2±2.8*

22.0±2.1*

28.6±0.6*

15.3±2.8*

20.8±0.4*

24.9±1.0*

46.2±5.1*

73.6±0.9*

77.1±4.6*

77.9±5.3*

Choroid plexus was incubated with [3H]PGD2 (30 nM) in the absence (control) or

presence of unlabeled compounds at 37ºC for 1 min. Each value represents the

mean±SEM (n=3). *p<0.01, significantly different from control.


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Table 2 Effect of several compounds on [3H]PGD2 uptake by Xenopus laevis

oocytes expressing mouse prostaglandin transporter (PGT/oocytes) and rat organic

anion transporter 3 (OAT3/oocytes).

Inhibitors Concentration

of inhibitor

(mM)

PGT/oocytes

% of control

OAT3/oocytes

% of control

Control

Prostaglandin D2 (PGD2)

Prostaglandin B1 (PGB1)

Bromocresolgreen

Benzylpenicillin

Dehydroepiandrosterone-3-sulfate

(DHEAS)

Probenecid

Estradiol-17β-glucuronide

(E217βG)

Indomethacin

p-Aminohippuric acid (PAH)

Diclofenac

L-Lactic acid

0.01

0.1

1

0.1

1

1

0.1

1

0.1

0.1

1

0.1

25

100±5

11.9±0.8

5.43±0.34*

6.50±0.30*

12.1±0.58*

8.13±0.37*

99.1±6.2

68.8±6.9*

82.3±5.5*

78.7±8.1*

ND

ND

ND

73.2±2.9*

100±5

28.2±2.4*

ND

ND

41.8±3.2*

48.9±1.4*

38.1±3.2*

65.5±8.0*

ND

ND

53.1±2.9*

82.5±13.0

38.6±1.8*

ND

[3H]PGD2 uptake by PGT/oocytes and OAT3/oocytes was measured at 20ºC for 1 h in

the absence (control) and presence of inhibitors at the indicated concentration. Each

value represents the mean±SEM (n=13-15). *p<0.01, significantly different from

control. ND, not determined.


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