Prostaglandin E2 metabolism in rat brain: Role of the blood-brain interfaces
Cerebrospinal Fluid Research
Prostaglandin E2 metabolism in rat brain: Role of the blood-brain interfaces
Eudeline Alix 1
Charlotte Schmitt 1
Nathalie Strazielle 0 1
Jean- Franois Ghersi-Egea 1
0 Brain-i , 34 Rue du Dr Bonhomme, Lyon, F69008 , France
1 INSERM, U 842, Lyon; Universite de Lyon; Faculte de medecine Laennec , UMR-S842, Lyon, F69372 , France
Background: Prostaglandin E2 (PGE2) is involved in the regulation of synaptic activity and plasticity, and in brain maturation. It is also an important mediator of the central response to inflammatory challenges. The aim of this study was to evaluate the ability of the tissues forming the blood-brain interfaces to act as signal termination sites for PGE2 by metabolic inactivation. Methods: The specific activity of 15-hydroxyprostaglandin dehydrogenase was measured in homogenates of microvessels, choroid plexuses and cerebral cortex isolated from postnatal and adult rat brain, and compared to the activity measured in peripheral organs which are established signal termination sites for prostaglandins. PGE2 metabolites produced ex vivo by choroid plexuses were identified and quantified by HPLC coupled to radiochemical detection. Results: The data confirmed the absence of metabolic activity in brain parenchyma, and showed that no detectable activity was associated with brain microvessels forming the blood-brain barrier. By contrast, 15-hydroxyprostaglandin dehydrogenase activity was measured in both fourth and lateral ventricle choroid plexuses from 2-day-old rats, albeit at a lower level than in lung or kidney. The activity was barely detectable in adult choroidal tissue. Metabolic profiles indicated that isolated choroid plexus has the ability to metabolize PGE2, mainly into 13,14-dihydro-15-ketoPGE2. In short-term incubations, this metabolite distributed in the tissue rather than in the external medium, suggesting its release in the choroidal stroma. Conclusion: The rat choroidal tissue has a significant ability to metabolize PGE2 during early postnatal life. This metabolic activity may participate in signal termination of centrally released PGE2 in the brain, or function as an enzymatic barrier acting to maintain PGE2 homeostasis in CSF during the critical early postnatal period of brain development.
Prostaglandin E2 (PGE2) is a main product of the
cyclooxygenase (Cox) pathway. Two Cox isoenzymes, Cox-1 and
Cox-2, convert arachidonic acid released by
phospholipases A2 to PGH2, which in turn is metabolized by
terminal prostaglandin E synthases into PGE2 . While Cox-1
is constitutively expressed in most tissues where it
finetunes physiological processes , Cox-2 expression is very
limited in normal conditions in peripheral organs. Yet it
is induced by inflammatory stimuli, and then, being
functionally coupled to microsomal prostaglandin E synthase
1, it plays a major role in the response to inflammation via
PGE2 production .
In the brain, our current understanding of PGE2 metabolic
cascade indicates that Cox-2 is constitutively expressed in
several neuronal cell populations, especially in
hippocampal and cortical glutamatergic neurons. The enzyme
participates in synaptic activity, hippocampal long-term
synaptic plasticity, and brain maturation (reviewed by
Chen and Bazan, and Minghetti [4,5]). Inflammatory
challenges trigger the cerebral upregulation of Cox 2,
particularly in venule endothelial cells, and the subsequent
production of PGE2. The prostaglandin acts as a
modulator of the sickness behavior syndrome, and specifically
induces fever via hypothalamic EP3 receptor activation
[6,7]. The efficiency and length of the biological response
to PGE2 is dependent upon the balance between its
production and its inactivation. In peripheral organs, the first
step of the inactivation process is mediated by
NAD+dependent 15-hydroxyprostaglandin dehydrogenase
(PGDH) . This enzyme is particularly active in the lung
or the kidney. By contrast, it is considered absent in the
brain of rodent and other mammalian species, from late
gestation throughout postnatal life [1,9,10]. PGE2
catabolism has however been reported specifically in the choroid
plexus of sheep during postnatal development and to
some extent in adulthood . Immunohistochemical
evidence for the presence of PGDH in lamb choroid
plexus exists also . The choroidal tissue constitutes a
major interface between the cerebrospinal fluid (CSF) and
the blood, and in conjunction with the cerebral capillaries
regulates the exchanges between the blood and the central
nervous system. The mechanisms responsible for this
crucial regulation are multiple and involve barrier and
transport, as well as metabolic processes towards biologically
active endogenous compounds.
We investigated in rats, whether the cells forming the
blood-brain interfaces are a site of PGE2 metabolism into
inactive compounds and as such, of signal termination.
We isolated cerebral capillaries and choroid plexuses from
developing and adult rat brain, measured PGDH activity
in these tissues, and identified the metabolites actually
produced from PGE2.
(NAD+) from Sigma (St Louis, MO, USA), bicyclo-PGE2
from Cayman Chemical (Ann Arbor, MI, USA), and
[3H]PGE2 (160 Ci/mmol) from Perkin Elmer Life Sciences
(Boston, MA, USA). Bovine serum albumin and dextran
used for capillary isolation were from I.D. Bio (Limoges,
France), and Sigma, respectively. All other reagents were
from high purity grades.
Animals and tissue isolation
Animal care and procedures have been conducted
according to the guidelines approved by the French Ethical
Committee (decree 87848) and by the European Community
directive 86609-EEC. Rats, 200240 g, Sprague-Dawley
males or timed pregnant females were obtained from
Harlan, Gannat, France. Following halothane anesthesia and
decapitation of the animals, rat brains were removed and
the choroid plexuses were sampled intact under a
stereomicroscope, briefly rinsed in Ringer-Hepes (RH) buffer
, and kept at -80C until used for enzymatic
measurement. Kidney, lung and meninges-free brain cortex were
also sampled. In some experiments freshly isolated intact
choroid plexuses from both adult and 2-day-old rats were
kept in RH buffer at 37C for metabolic analysis.
Microvessels from 9-day-old and adult brain cortices were
isolated at 4C in oxygenated buffers according to a
previously described procedure , except that the
capillaries were collected on a 40 m-mesh nylon filter instead
of glass beads. The purity of each preparation was
controlled by phase contrast microscopy and by measuring the
glutamyl transferase specific activity as a capillary marker
Prostaglandin dehydrogenase activity measurement
Pools of choroid plexuses from at least eight 2-day-old or
four adult animals, pools of isolated brain microvessels
from twelve 9-day-old animals or four adults, brain
cortex, kidney or lung tissue were homogenized in 50 mM
Tris, 1 mM EDTA, 2 mM DTT buffer, pH 7.4, using a
glassglass homogeniser. The homogenates were centrifuged for
30 min at 14 000 rpm at 4C, and the resulting
supernatant assayed for PGDH activity. This measurement was
performed at 37C by kinetic analysis on a VARIAN Carry
100 double-beam spectrophometer (Mulgrave, VIC,
Australia) set at 340 nm as follows: the supernatant was
added to the Tris-EDTA-DTT buffer in both reference and
sample cuvettes. After baseline stabilisation, NAD+ (1
mM) was added to both cuvettes and the baseline further
recorded until it stabilized again. PGE2 (20 M) was then
added to the sample cuvette and the optical density was
recorded to follow the appearance of the reduced
nucleotide NADH. The specific activity was calculated using the
extinction coefficient of 6.22 10-3 M-1.cm-1. An aliquot
of kidney supernatant was run in each set of
measurements as an internal control. The total protein content of
the supernatants was determined by the method of
Peterson  with bovine serum albumin as the standard.
PGE2 metabolism by isolated choroid plexuses
Choroid plexuses from lateral and fourth ventricles were
treated separately. Choroid plexuses from four 2-day-old
animals or two adult animals were pooled and incubated
on a rotating shaker in 160 l of RH at 37C for 5 or 45
min in the presence of 100 nCi of [3H]PGE2. Incubation
medium without tissue was run in parallel with each set
of measurements. At the end of the incubation, the
choroidal tissue was removed and added to 40 l of
distilled water, homogenized in the presence of an
additional 40 l of acetonitrile, and centrifuged at 14,000 rpm
for 30 min. The resulting supernatant and the incubation
medium were then analyzed by HPLC. Choroid plexus
protein content was evaluated separately on pools of
choroid plexus tissue from the same litter (2-day-old
animals), or from the same batch of animals (adults).
HPLC analysis, and expression of the results
Incubation media and homogenate supernatants were
analysed by reverse phase HPLC performed on a LC10
Shimadzu system (Duisburg, Germany) as follows:
Samples (20 or 40 l) were loaded with a mix of unlabelled
PGE2 and its metabolites to allow UV detection, applied
onto an Ultrasphere ODS RP-18 analytical column (5 m,
46 mm 150 mm, Beckman, Fullerton, California, USA),
and eluted using a mobile phase of 35% acetonitrile/0.1%
acetic acid/water pumped at 1 ml/min. Absorbance of the
effluent was monitored at 210 nm. The effluent was
collected for radiochemical analysis by liquid scintillation
counting. Retention times of PGE2, 15-keto-PGE2,
13,14dihydro-15-keto-PGE2 and bicyclo-PGE2 were 8.5, 12, 16
and 43 min, respectively. The purity of radiolabelled PGE2
was estimated from the incubation medium without
choroidal tissue, as the ratio of radioactivity associated
with PGE2 to the total radioactivity recovered and was
taken into account in further calculations. Amounts of
remaining PGE2 and of the metabolites produced during
the incubation with the choroidal tissue were expressed as
percentage values of the initial PGE2-associated
radioactivity. The radioactive profile obtained from the
incubation medium without tissue was used as a background
profile. The background radioactivity eluted within the
time-frame of collection for each metabolite was
subtracted from the corresponding radioactivity measured in
incubation medium following tissue metabolism. The
amount of radioactivity (nCi) associated with PGE2 and
each metabolite was calculated separately for the medium
and the choroidal tissue, and then summed to generate
the total % of PGE2 remaining, or metabolite produced at
the end of the incubation period. To establish the
tissuemedium distribution of PGE2 and
13,14-dihydro-15keto-PGE2, the amount of each species present in the
incubation medium and in the choroidal tissue at the end of
the incubation was expressed as % of the total amount
(medium plus tissue).
15-hydroxyprostaglandin dehydrogenase specific activity
at blood-brain interfaces
PGDH activity was measured in homogenates of choroid
plexuses, brain microvessels and cortical tissue (Table 1).
The enzymatic analysis confirmed the absence of a
detectable PGE2 metabolism in cerebral cortex of both adult and
2-day-old animals, and indicated that adult cerebral
microvessel preparations also lack PGDH activity. The
earliest postnatal period allowing pure capillary isolation is
day 9 which marks the end of the sprouting period during
which the basal lamina of primitive vessels remains thin
and uneven . The activity measured in microvessel
preparations from 9-day-old animals was also
undetectable (not listed in Table 1). By contrast, this enzymatic
activity was readily detected in choroidal material
sampled from lateral or fourth ventricle of 2-day-old rat brain.
In both types of choroid plexus the activity was lower than
in kidney and lung, two peripheral organs involved in
prostaglandin signal termination (p < 0.01 and p < 0.05,
respectively, one-way ANOVA followed by
TukeyKramer's test). It strongly decreased in adult choroid
plexus (p < 0.05, one-tailed student's t-test for unequal
PGE2 metabolism in choroid plexuses
To investigate further the ability of the choroidal tissue to
inactivate PGE2, we analyzed the metabolites produced
upon exposure of intact isolated choroid plexus to the
prostaglandin. Radiolabelled PGE2 was used as substrate
and the metabolites were quantified by HPLC coupled to
radiochemical detection. Figures 1a and 1b show the
spectrophotometric profiles obtained using incubation
medium supplemented or not, with a mix of unlabelled
PGE2 and its main metabolites. The chromatographic
conditions were adequate to separate the two potential PGE2
metabolites, i.e. 15-keto-PGE2 and
Data (in nmol.mg protein-1.min-1) are expressed as mean SE (n).
ND: not detectable. na: not available (see text). The detection limit
was calculated as being 0.02 for microvessel and choroidal
preparations, and 0.01 for cerebral cortex.
keto-PGE2, as well as a non enzymatic cyclization
breakdown product of 13,14-dihydro-15-keto-PGE2 known as
bicyclo-PGE2 . Typical radioactive profiles obtained
by incubating [3H]PGE2 in the absence or presence of
isolated intact lateral ventricle choroid plexuses of 2-day-old
rats are shown in Figure 1c and 1d, respectively. In
addition to the above-mentioned metabolites, an additional
peak appeared with a short retention time (Peak 1; 1.75
min). Because the biotransformation of PGE2 into
15keto-metabolites led to the release, as tritiated water, of
one of the seven tritiated hydrogen atom on the
prostaglandin, it is likely that tritiated water contributed to the
radioactivity in this peak.
The quantitative analysis of the data indicates that after 5
min incubation with lateral ventricle choroid plexuses
from four 2-day-old animals, 12% of initial PGE2 has
been metabolized, (representing a metabolic clearance of
18 l, Figure 2). The most abundant metabolite,
13,14dihydro-15-keto-PGE2 represents 9% of the radioactivity
initially associated with PGE2. When the incubation time
was extended to 45 min to allow substrate recycling, thus
resulting in the biotransformation of 66% of PGE2,
13,14dihydro-15-keto-PGE2 remained the main metabolite.
The radioactivity associated with 15-keto-PGE2 and
bicyclo-PGE2 at 45 min represented only 0.13 and 1% of the
total radioactivity initially associated with PGE2 (Figure
2). At both 5 and 45 min, 75% of the amount of
radioactivity forming Peak 1 may be accounted for by tritiated
water formed during the transformation of PGE2 into
Very similar data were obtained using choroid plexuses
sampled from the cerebral fourth ventricle of 2-day-old
animals (not shown). The ability of intact isolated
choroid plexuses from adult animals to inactivate PGE2,
was also studied and compared to that of 2-day-old
animals. The total amounts of PGE2 metabolized by lateral
and fourth ventricle choroid plexuses of adults were
respectively 10.5 and 6% of those biotransformed by the
corresponding choroidal tissue of 2-day-old rats,
following normalization for total protein content. As in
2-dayold material, the main metabolite identified was
13,14dihydro-15-keto-PGE2 (data not shown).
Location of PGE2 metabolite secretion by choroid plexuses
The medium and the choroidal tissue were analyzed
separately to provide insight into the site of metabolite
excretion from choroid plexus cells. The data are shown for
lateral ventricle choroid plexuses (Figure 3). PGE2
remaining at the end of the incubation period of 5 min was
mostly found in the incubation medium. By contrast,
13,14-dihydro-15-keto-PGE2 was mostly associated with
the choroidal tissue, i.e. remained intracellular or
concentrated within the stroma of the choroid plexus, rather than
lRFisiamgduiobrcyehie1somlaictaeldrechveorrsoeidphpalesxeuHsePsLC analysis of PGE2
metaboRadiochemical reverse phase HPLC analysis of PGE2
metabolism by isolated choroid plexuses. Typical
chromatograms are shown: a and b are UV profiles of RH buffer
(a) and RH buffer supplemented with PGE2 and its main
potential metabolites (b); c and d are radiochemical
chromatograms of RH medium incubated for 45 min in the absence
(c) and presence (d) of lateral ventricle choroid plexuses
from 2-day-old rats. In c and d the y axis represents
radioactivity concentration (dpm/ml) in each collected fraction.
KPGE2 DHKPGE2 BCPGE2
vPFeGignEturrimcele2staobfo2li-sdmayb-yolidsorlatebdracihnoroid plexuses from lateral
PGE2 metabolism by isolated choroid plexuses from
lateral ventricles of 2-day-old rat brain. Data are
expressed as mean SD of three different experiments, and
represent the amount of radioactivity associated to each
molecular species, relative to the initial radioactivity
associated to PGE2 (see method), after either 5 or 45 minutes of
incubation. * and **: different from PGE2 amount in
incubation without choroidal tissue, p < 0.05 and 0.01, respectively,
paired student's t-test. Abbreviations: KPGE2: 15-keto-PGE2,
DHKPGE2: 13,14-dihydro-15-keto-PGE2, BCPGE2:
being excreted at the apical membrane of the epithelial
cells. After 45 min, most of this metabolite was measured
in the medium. The small amount of intermediate
metabolite 15-keto-PGE2 produced remained associated with
the tissue (not shown). Very similar data were obtained
for fourth ventricle choroid plexus (not shown).
In this paper, we explored PGE2 catabolism in rat brain,
focusing more specifically on the involvement of the
blood-brain interfaces, at both postnatal and adult stages.
PGDH is considered as a key oxidizing enzyme in PGE2
inactivation cascade, as the primary metabolite
15-ketoPGE2has a greatly reduced biological activity.
13,14-dihydro-15-keto-PGE2 is a secondary metabolite without
biological activity generated by 1315-keto-prostaglandin
In agreement with other groups, we observed no PGDH
activity in brain cortical tissue in either young or adult
animals, and we showed the absence of detectable enzyme in
microvessels, i.e. at the blood-brain barrier. By contrast,
and in accordance with data presented in sheep , we
gathered evidence for PGE2 catabolic activity at the
choroid plexus, which is the main site of the blood-CSF
barrier. First, a significant specific activity of PGDH was
measured in choroidal tissue homogenates prepared from
2-day-old rats. Second, the incubation of isolated whole
Tissue-medium distribution of PGE2 and
13,14-dihydro-15-keto-PGE2 following exposure of 2-day-old rat
lateral ventricle choroid plexuses to PGE2. PGE2 and
13,14-dihydro-15-keto-PGE2 were quantified separately in
the medium and in the choroidal tissue. The data are
expressed as percentage of the total amount of molecule
(either remaining PGE2 or produced
13,14-dihydro-15-ketoPGE2), at the end of the 5- and 45-minute incubation period.
Mean SD, n = 3. Abbreviations as in Fig 2.
choroid plexuses with PGE2, coupled to HPLC analysis,
demonstrated the production of PGE2 metabolites, in
particular 13,14-dihydro-15-keto-PGE2, thereby revealing the
functional coupling of 1315-keto-prostaglandin
reductase to PGDH in the choroidal tissue.
The functional significance of this choroidal metabolic
pathway may relate either to the termination of
CSFborne PGE2 signal, or to the prevention of blood-borne
PGE2 penetration into the CSF. In our experimental
setting for ex-vivo choroid plexus incubation, the isolated
tissues were kept entire, which maximally limits rapid
transfer between the external medium and the choroidal
stromal core, and allows us to assume that most PGE2 was
presented apically, i.e. at the CSF-side of the choroidal
epithelium. Our results therefore suggest that in vivo,
centrally released PGE2 that circulates in CSF will be
metabolized to some extent by choroidal metabolizing enzymes.
In line with this, an apical uptake of PGE2 mediated by an
inwardly-directed probenecid-sensitive transport system
has been reported in choroidal epithelium of different
species [11,18,19]. Both metabolic and transport affinity
constants have been determined in the micromolar range
[8,20]. Given that PGE2 levels in the CSF remain below
this concentration in physiological as well as pathological
conditions [21-23], neither the enzymatic nor the
transport process will reach saturation. The extent to which
choroidal PGE2 metabolism can impact on the
concentration of CSF-borne PGE2 remains however to be evaluated.
Although PGDH activity is readily detected in choroidal
tissue from young pups, it is 10 to 20 times lower than in
kidney, or lung which is the main organ involved in
peripheral signal termination of circulating
prostaglandins . In ex-vivo tissues from 2-day-old animals, after
5min incubations, a significant amount of untransformed
PGE2, similar to the total amount of metabolites
produced, was found associated with the choroidal tissue.
Although the precise localization of PGDH (epithelial
and/or stromal) within the CP is unclear (see infra), the
latter observation suggests that the metabolism capacity
of the tissue towards PGE2 is lower than its uptake
capacity. Transepithelial flux of PGE2 has been demonstrated in
an in vitro model of the choroidal epithelium, implying
that following its apical uptake from the CSF, native PGE2
can be exported across the basolateral stroma-facing
membrane . The relative capacity of this basolateral
efflux mechanism, favoring PGE2 elimination in blood
and thus supplementing the enzymatic signal termination
mechanism needs to be established by comparison to
uptake and metabolism in order to delineate how
CSFborne PGE2 concentration is controlled in developing
PGE2 catabolism in the choroidal tissue may also be
relevant in preventing blood-borne PGE2 from entering the
CSF during early postnatal life. During this period, PGE2
is associated with hypothalamic maturation processes
 and an increase in CSF PGE2 induces respiratory
depression [reviewed in [11,12]]. Therefore abnormal
blood PGE2 concentrations following infection or
inflammation, were they to disrupt physiological PGE2 levels in
CSF, could possibly lead to cerebral dysfunction. Based on
the directionality and membrane distribution of the
epithelial organic anion transporters that are likely
candidates for membrane transfer of the prostaglandin ,
blood-to-CSF permeability to PGE2 is expected to be
much lower than CSF-to-blood permeability. In 2-day-old
rats, the metabolic activity of the choroid plexus tissue
towards the prostaglandin will add an enzymatic barrier
component to the transporter-mediated barrier properties
of the epithelium, thereby contributing to buffer blood
perturbations and maintain PGE2 homeostasis in CSF
during this critical period of life. Of note, ontogenic
maturation of the choroid plexuses is precocious and this tissue
appears to play key functions in the control of brain
homeostasis when the cerebral vasculature is still
developing. [25,26]. The metabolic capacity displayed by the
choroid plexuses towards PGE2, during the postnatal
period highlights the early functional maturity of the
In the adult, PGDH enzymatic activity is strongly
decreased in choroidal tissue, a finding confirmed by the
age-dependent decreased metabolic capacity observed in
isolated choroid plexus. This leaves peripheral organs
such as lung as the most likely sites of catabolism for the
prostaglandin following its clearance from adult brain .
In ex vivo studies using short duration incubation, PGE2
metabolites were mostly associated with the choroidal
tissue, indicating that they were produced by the epithelial
cells and then preferentially released in the stroma, and/
or produced by the fibroblasts or other stromal cells.
When the incubation was prolonged,
13,14-dihydro-15keto-PGE2 reached the external medium, probably as a
result of diffusion from stroma. We previously showed,
using a polarized cellular model of the blood-CSF barrier,
that 13,14-dihydro-15-keto-PGE2 was produced and
excreted at the basolateral membrane of the epithelial
cells . The latter step may involve the multidrug
resistance associated protein abcc4, which transports organic
anions such as prostaglandins , although its affinity
for keto-metabolites remains to be established. This
transporter has been immunodetected at the basolateral
membrane of choroidal epithelium in several species .
PGE2 metabolism was however limited in the epithelial
cells and did not significantly impede the transcellular
flux of the prostaglandin . Alternatively, the stromal
hypothesis of PGE2 metabolism is supported by the
immunohistochemical description in sheep of a switch in
PGDH localisation from the epithelium to the stromal
cells at birth . In rat, attempts to locate PGDH in
choroidal tissue by immunohistochemistry in our
laboratory have been so far inconclusive (not shown).
Regardless of the cellular site of PGE2 metabolism, the stromal
i.e. blood side of metabolite excretion adds to the
efficiency of the metabolic barrier by driving the clearance of
the metabolite towards the blood circulation.
Metabolism of PGE2 occurs in the rat choroid plexus tissue
at early postnatal stage, leading to the production of the
inactive 13,14-dihydro-15-keto-PGE2 metabolite. This
function of the choroidal tissue, which disappears in
adulthood, may be involved in maintaining CSF PGE2
homeostasis during the critical early phase of postnatal
The authors declare that they have no competing interests.
Data collection was performed by EA and CS. The study
was conceived, designed and funded by JFGE and NS. NS
realized the microdissections and helped to draft and
revise the manuscript, and JFGE finalized the manuscript,
and performed the statistical analyses.
This work was realized with the support of ARSEP.
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