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(Received for publication, May 3, 1995; and in revised form, July 5,
1995) From the
The overall goal of this study was to determine the mechanisms
by which nucleobases are transported in the choroid plexus. Choroid
plexus tissue slices were obtained from the lateral ventricles of
rabbit brains and depleted of ATP with 2,4-dinitrophenol. In the
presence of an initial inwardly directed Na
Nucleobases and their structural analogs have become important
drugs in the treatment of a number of viral infections of the central
nervous system such as cytomegalovirus retinitis, herpes simplex
encephalitis, and AIDS-related dementia complex. Therefore, nucleobase
transport mechanisms at the major barriers of the central nervous
system, namely the blood-brain barrier and the blood-cerebrospinal
fluid barrier (choroid plexus epithelium), may play a critical role in
targeting nucleobase analogs to the affected tissues. Moreover, such
transport systems may be important in the salvage of purines for
nucleic acid synthesis in the brain. Saturable, low affinity
nucleobase transporters have been identified in the blood-brain
barrier(1, 2) . However, it appears that these
transporters may not be sufficient to mediate the flux of important
quantities of hypoxanthine, the principal salvageable nucleobase, or
other nucleobases into the brain. As a result, attention has focused on
the choroid plexus as a possible route of entry for hypoxanthine and
other nucleobases into the brain(3) . In mammalian cells,
transcellular flux of nucleobases is mediated by specific transporters
in the plasma membranes. Two major classes of nucleobase transporters
have been characterized: equilibrative and concentrative. Equilibrative
transporters are present in a number of cell types and are broadly
selective for purine and pyrimidine nucleobases. Recent studies with
acyclovir and ganciclovir, nucleobase/nucleoside analogs, demonstrate
that these agents permeate the human erythrocyte by way of
equilibrative mechanisms(14, 15) . Recently,
concentrative nucleobase transporters have been identified in
LLC-PK The goal of
this study was to determine the mechanisms of nucleobase transport in
the choroid plexus. Our data demonstrate the presence of a novel
Na
Thin
layer chromatography methods were used to determine whether metabolism
or degradation of hypoxanthine had occurred. Procedures used were as
described previously(10) .
Statistical analysis was carried out by a Student's
unpaired t test. A probability, p, of less than 0.05
was considered significant. Data points were determined in triplicate
for each experiment. Data, unless mentioned otherwise, are expressed as
the mean ± S.E. of data obtained from three experiments in
choroid plexus tissue from separate animals. Standard methods were
used to determine IC To determine the stoichiometric coupling
between Na
where a is V
In the absence of a Na
Figure 1:
Hypoxanthine uptake (0.24
µM) in ATP-depleted rabbit choroid plexus tissue slices.
The uptake of hypoxanthine (V
In contrast, in the presence of an initial inwardly directed
Na Kinetic
experiments (Fig. 2) were performed in which the rate of
hypoxanthine uptake (at 30 s) as a function of concentration was
determined in the presence of an inwardly directed Na
Figure 2:
The rate of Na
To determine the stoichiometry (Fig. 3) of the
Na
Figure 3:
The initial rate of uptake of hypoxanthine
(30 s) in ATP-depleted rabbit choroid plexus tissue slices as a
function of Na
The effect of various purines on
Na
Figure 4:
The effect of purines (100
µM) on hypoxanthine (0.24 µM) uptake (at 30
s) in ATP-depleted rabbit choroid plexus tissue slices. Hypoxanthine (Hypo), adenine, adenosine, dideoxyadenosine (DDA),
guanine, and xanthine significantly (p < 0.05) inhibited
Na
Figure 5:
The effect of pyrimidines (100
µM) on hypoxanthine (0.24 µM) uptake (at 30
s) in ATP-depleted rabbit choroid plexus tissue slices. Cytosine,
thymine, and uracil significantly (p < 0.05) inhibited
Na
Figure 6:
The effect of increasing concentrations of
xanthine on Na
The
effect of the nucleobases, hypoxanthine and thymine, on the
Na
Figure 7:
The effect of nucleobases on
Na
Previous studies demonstrated that hypoxanthine accumulates
and is metabolized in rabbit choroid plexus tissue slices(3) .
However, the driving force of the transport mechanism(s) was not
determined. By using ATP-depleted choroid plexus slices, we
experimentally imposed an initial inwardly directed Na The data in this study suggest
that the Na Second, the
substrate selectivity of the Na Our data are consistent with a 2:1
coupling ratio for Na The Na In
conclusion, a Na
Volume 270,
Number 39,
Issue of September 29, pp. 22816-22819, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR A Na-DEPENDENT NUCLEOBASE TRANSPORTER
WITH BROAD SUBSTRATE SELECTIVITY (*)
gradient,
hypoxanthine accumulated in the tissue slices against a concentration
gradient. Na
-stimulated hypoxanthine uptake was
saturable with a K
of 31.1 ± 9.71
µM and a V
of 2.69 ± 0.941
nmol/g/s (mean ± S.E.). Na-stimulated
hypoxanthine uptake was inhibited by (100) µM naturally
occurring purine and pyrimidine nucleobases (adenine, cytosine,
guanine, hypoxanthine, thymine, uracil, and xanthine) as well as by the
nucleoside analog, dideoxyadenosine. The stoichiometric coupling ratio
between Na
and hypoxanthine was 1.7:1. The data
demonstrate the presence of a novel Na
-dependent
nucleobase transporter in the choroid plexus, which is distinct from
the previously described Na
-nucleoside transporter in
choroid plexus and from Na
-dependent nucleobase
transporters in other tissues in terms of its kinetics, substrate
selectivity, and Na
-nucleobase stoichiometry. This
transporter may play a role in the targeting of both salvageable
nucleobases and therapeutic nucleoside analogs to the central nervous
system.
cells(4) , guinea pig
kidney(5, 13) , rat jejunal tissue(6) , and
guinea pig placenta(7) . These transporters are secondary
active and Na-dependent and mediate the influx of
specific nucleobases. The substrate selectivity of the transporter in
LLC-PK
cells has been studied in considerable detail and
includes selected purine and pyrimidine nucleobases.-dependent nucleobase transporter in the choroid
plexus, which is distinct from previously described
Na
-driven nucleobase transporters in other tissues in
terms of its substrate selectivity and kinetics. This transporter may
play an important role in the delivery of both salvageable and
therapeutic nucleobases to the central nervous system.
Preparation of ATP-depleted Choroid Plexus Tissue
Slices
Choroid plexus tissue slices from rabbit were
ATP-depleted by the method of Whittico(8) . Choroid plexuses
were obtained from the lateral ventricles of New Zealand White rabbits.
The choroid plexus tissue was placed in KCl buffer (37 °C) of the
following composition: KCl (150 mM), mannitol (40
mM), and HEPES (25 mM), pH 7.4, with 1 M Tris. Choroid plexus tissue was cut into 2-3-mm pieces and
ATP-depleted by incubating at 37 °C for 20 min in 2,4-dinitrophenol
(250 µM) in KCl buffer. Under these conditions, the ATP
concentration is reduced to less than 10% of control(9) .
Following this incubation, the choroid plexus slices in buffer were
stored on ice until uptake experiments were performed.N-Ethylmaleimide (NEM)
ATP-depleted choroid plexus slices were
incubated at room temperature in NEM (1 mM) for 45 min as
described previously(11) .IncubationAccumulation Studies
Uptake of
[
H]hypoxanthine was studied by methods published
previously(10) . Briefly, individual choroid plexus tissue
slices were incubated with 140 µl of reaction mixture containing
[H]hypoxanthine (0.24 µM),
[
C]mannitol (25.4 µM), and
2,4-dinitrophenol (250 µM) in KCl (150 mM) or
NaCl buffer (150 mM). The uptake was stopped by blotting on
laboratory tissue. The choroid plexus tissue slices were then dried and
weighed. Tissue slices were dissolved. H and C
were determined in the dissolved tissue samples and the corresponding
reaction mixture by dual isotope liquid scintillation counting.Data Analysis
The radioactive content from each
choroid plexus tissue slice was expressed as a volume of distribution (V
) as described
previously(8, 10) .
values and Michaelis-Menten
kinetics(10) .
and hypoxanthine, a modified version of the
Hill equation was used,
/K
when K
C
in the reaction
mixture. The data were transformed with a logarithm and linearly
regressed to obtain a and n. This equation has been
used previously in stoichiometry studies(12) .
Materials
[H]Hypoxanthine
(11.6 Ci/mmol), [H]thymidine (65 Ci/mmol), and
[C]mannitol (56 mCi/mmol) were purchased from
either Amersham Life Science, Arlington Heights, IL or Moravek
Biochemicals, Inc., Brea, CA. Hypoxanthine, adenine, adenosine,
dideoxyadenosine (ddA), guanine, xanthine, caffeine, cytosine, thymine,
uracil, thymidine, and proline were purchased from either Sigma or
Aldrich. All other chemicals were purchased from either Sigma, Fisher
Scientific, or Aldrich. New Zealand White rabbits were purchased from
Nitabell Rabbitry, Hayward, CA. Cytoscint ES scintillation
fluid was purchased from ICN Biomedical Inc.
gradient,
hypoxanthine accumulated in the tissue slices and reached an
equilibrium (V
= 2.57 ± 0.29) in
approximately 5 min (Fig. 1). This uptake was not reduced by any
compound (including unlabeled hypoxanthine) in the concentration range
used in these studies. These results suggest that in the absence of a
Na gradient, the uptake of hypoxanthine represents a
nonselective or low affinity binding process or transport process.
) was
examined in the absence (squares) and presence (circles) of an initial inwardly directed Na
gradient. Each data point represents the uptake of hypoxanthine
(mean ± S.E.) from three
experiments.
gradient (150 mM), hypoxanthine
accumulated temporarily (``overshoot phenomenon'') in the
tissue slices above the equilibrium value (V
= 6.11 ± 0.91 at 1 min) (Fig. 1). Thin layer
chromatography (TLC) studies indicated that hypoxanthine was not
significantly metabolized at 30 s or 5 min (data not shown). gradient (150 mM). The data are consistent with a single
saturable process. The data from each of the three experiments were fit
to an appropriate Michaelis-Menten equation, which included a linear
component(8) . The K
and V (mean ± S.E.) for
Na-stimulated hypoxanthine uptake were 31.1 ±
9.71 µM and 2.69 ± 0.94 nmol/g/s, respectively.
-stimulated
hypoxanthine uptake (at 30 s) in ATP-depleted rabbit choroid plexus
tissue slices as a function of hypoxanthine concentration. Points
represent the data obtained in a representative experiment. Mean
(± S.E.) K
and V
values were determined from three separate experiments. The curve represents the best fit to the Michaelis-Menten
equation. In this experiment, the K was
44.1 µM and the V
was 4.44
nmol/g/s.
-dependent nucleobase transport system, the
Na
-dependent uptake of hypoxanthine (0.24
µM) was examined in the presence of increasing
Na
concentrations (0-140 mM). The
uptake of hypoxanthine (V
) was sensitive to
Na concentration. The data were fit to a Hill equation
as described under ``Experimental Procedures.'' The Hill
coefficient was 1.7 ± 0.4 (mean ± S.E.) for
Na
:hypoxanthine and was significantly different from 1
but not from 2.
concentration. Points represent the mean (± S.E.) of data obtained in three
separate experiments. The curve represents the best fit to the
Hill equation. A Hill coefficient of 1.7 ± 0.4 was
obtained.
-stimulated hypoxanthine uptake was examined (Fig. 4). At concentrations of 100 µM,
hypoxanthine, adenine, adenosine, ddA, guanine, and xanthine
significantly inhibited Na
-dependent hypoxanthine
uptake (V
) at 30 s (p < 0.05).
Hypoxanthine uptake was slightly inhibited by 2`-deoxyadenosine (29%)
but not 3`-deoxyadenosine (data not shown). Caffeine (100
µM) did not significantly inhibit
Na-dependent hypoxanthine transport. At concentrations
of 100 µM, the pyrimidine nucleobases, cytosine, thymine,
and uracil, significantly inhibited Na
-dependent
hypoxanthine uptake at 30 s (p < 0.05) whereas the
pyrimidine nucleoside, cytidine, did not (Fig. 5). The amino
acid, proline, also did not inhibit Na
-dependent
hypoxanthine uptake (data not shown). A range of concentrations
(0-1 mm) was used to determine IC
values for
xanthine, uracil, and adenine. Xanthine (65.7 ± 5
µM) (Fig. 6), uracil (64.6 ± 11.2
µM), and adenine (77.9 µM) were potent
inhibitors (IC
values in the low micromolar range) of
Na
-dependent hypoxanthine transport. Consistent with a
single transport mechanism, inhibition curves were monophasic.
-dependent hypoxanthine uptake. Solid and hatchedbars represent data obtained in rabbit
choroid plexus slices in the presence and absence of an inwardly
directed Na
gradient, respectively. Bars represent the mean (± S.E.) of data obtained in three
separate experiments.
-dependent hypoxanthine uptake. Solid and hatchedbars represent data obtained in rabbit
choroid plexus slices in the presence and absence of an inwardly
directed Na
gradient, respectively. Bars represent the mean (± S.E.) of data obtained in three
separate experiments.
-hypoxanthine uptake. Points represent the data obtained in a representative experiment. Mean
(± S.E.) IC
values were determined from three
separate experiments. In this experiment, the IC
was 59.6
µM.
-dependent uptake of the nucleoside, thymidine, was
examined (Fig. 7). At concentrations of 100 µM,
hypoxanthine and thymine did not significantly inhibit
Na
-dependent thymidine uptake at 30 s. These data
suggest that nucleobases and nucleosides do not share the same
Na
-stimulated transporter in the choroid plexus.
Further studies using NEM, an irreversible sulfhydryl modifying agent,
significantly inhibited Na
-thymidine uptake but not
Na
-hypoxanthine uptake (data not shown). This further
demonstrates that these two compounds do not share the same
transporter.
-nucleoside transport in ATP-depleted rabbit choroid
plexus tissue slices. [
H]Thymidine (6
µM) uptake (30 s) was determined in ATP-depleted rabbit
choroid plexus tissue slices in the presence of unlabeled thymidine,
thymine, or hypoxanthine (100 µM) and an inwardly directed
Na gradient. Bars represent the mean
(± S.E.) of data obtained in three separate experiments. Solid and hatchedbars represent data in the
presence and absence of an inwardly directed Na
gradient, respectively. Only unlabeled thymidine significantly (p < 0.05) inhibited Na
-dependent
thymidine uptake.
gradient and directly demonstrated that hypoxanthine transport is
coupled to a Na
gradient (Fig. 1). These data
provide the first demonstration of a Na
-nucleobase
transporter in the choroid plexus.
-dependent hypoxanthine transporter in the
choroid plexus is distinct from Na
-dependent
hypoxanthine transporters in other tissues. First,
Na
-dependent hypoxanthine uptake in the choroid plexus
has a lower affinity (31.1 µM) than
Na
-stimulated hypoxanthine uptake in guinea pig kidney
(4.4 µM) (5, 13) and LLC-PK
cells (0.79 µM) (4) but a higher affinity
than the saturable uptake system for hypoxanthine (400 µM)
identified in the blood brain barrier(2) .-stimulated
hypoxanthine transporter in the choroid plexus differs from that of
Na
-stimulated hypoxanthine transporters in other
tissues. In the choroid plexus, the purine nucleobases, adenine,
guanine, and xanthine, and the pyrimidine nucleobases, cytosine,
thymine, and uracil, were potent inhibitors of
Na
-dependent hypoxanthine uptake ( Fig. 4and Fig. 5). In contrast, in LLC-PK
cells the
nucleobases, adenine, cytosine, and xanthine, did not inhibit
Na-stimulated hypoxanthine uptake(4) . The
transporter also appears to differ in its substrate selectivity from
the Na
-dependent nucleobase transporter in renal brush
border membrane vesicles from guinea pig, which excludes
adenine(5, 13) . A Na
-dependent
nucleobase transporter for pyrimidines in rat jejunal tissue has been
characterized(6) ; however, the interactions of purines with
the transporter were not examined. The interaction of ddA with the
Na
-dependent nucleobase transporter suggests that the
choroid plexus may play a role in the transport of certain clinically
relevant nucleoside analogs.
-hypoxanthine transport.
Na
-hypoxanthine transport exhibits a 1:1 stoichiometry
in LLC-PK
cells and a 2:1 stoichiometry in the guinea pig
kidney(13) . Similar data have been obtained for
Na-nucleoside transport in choroid plexus. That is, a
2:1 Na
-nucleoside stoichiometry has been obtained for
the N-3, Na
-nucleoside transporter in choroid
plexus(10) .
-nucleobase transporter
in the choroid plexus also appears to be distinct from the
Na
-nucleoside transporter in the same tissue. Neither
cytidine (Fig. 5) nor thymidine (data not shown) inhibited
Na
-stimulated hypoxanthine uptake in the choroid
plexus. Conversely, neither hypoxanthine (100 µM) nor
thymine (100 µM) inhibited Na
-stimulated
thymidine uptake (Fig. 7). Moreover, previous studies in this
laboratory have demonstrated that the sulfhydryl modifier, NEM,
irreversibly inhibits Na
-nucleoside transport in
choroid plexus(11) . However, NEM did not inhibit
Na
-hypoxanthine uptake in rabbit choroid plexus.
-dependent nucleobase transporter in
choroid plexus from rabbit has been characterized. This transporter is
broadly selective for both purine and pyrimidine nucleobases and
appears to differ from the recently characterized
Na
-nucleobase transporters in guinea pig kidney and
LLC-PK
based on its kinetics and substrate selectivity.
However, this cannot be confirmed until the transport has been cloned
and sequenced. Further studies are also needed to determine the
relative contribution of the choroid plexus (in comparison with the
blood brain barrier) in transporting physiologically relevant
quantities of nucleobases into the brain and the role of the
transporter in targeting nucleobase and nucleoside analogs to the
central nervous system.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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