(Received for publication, December 1, 1995; and in revised form, February 21, 1996)
From the
Our laboratory previously has shown apparent carrier-mediated
glutathione (GSH) uptake across the blood-brain barrier (BBB) in two
animal models. In the present study, when Xenopus oocytes were
injected with bovine brain capillary mRNA expression of intact GSH,
uptake was observed after 3 days. When total mRNA was converted to cDNA
and subfractionated with subsequent cRNA injection into oocytes, three
distinct fractions (5, 7-8, and 11-12) expressed
carrier-mediated intact GSH transport. Northern blot analysis
established the presence of RcGshT, the previously cloned
sodium-independent hepatic canalicular transporter, only in fraction 5.
GSH transport activity in fraction 7 was significantly inhibited by
replacement of NaCl with choline chloride and by
sulfobromophthalein-GSH, neither of which affects RcGshT. The
Na-dependent GSH uptake kinetics exhibited high
affinity (
400 µM) and low affinity (
10
mM) components. Fraction 11 expressed
Na
-independent transport of intact GSH and also
contained the GGT transcript. In conclusion, we have identified three
distinct sized transcripts from bovine brain capillary mRNA which
express GSH transport: one fraction expresses a novel
Na
-dependent GSH uptake which can be dissociated
unequivocally from both GGT and RcGshT for the first time and which may
account for uptake of GSH against its electrochemical gradient at the
BBB.
Inherited disorders of GSH metabolism cause severe neurological
defects. GSH is a key factor in defense against hydrogen peroxide and
other reactive metabolites and also may act as a
neuromodulator(1, 2, 3) . GSH is present in
whole brain and in brain endothelium in millimolar levels(4) .
Endothelial cells, constituting the blood brain barrier (BBB), ()play a vital role in overall GSH metabolism, although the
mechanisms are not clearly understood, especially with respect to the
potential role of uptake and the responsible carriers. We recently
obtained evidence that GSH transport is present in the BBB in the rat
and in the perfused guinea pig brain, which is developmentally
regulated(5, 6, 7) . This transport system
differed in substrate specificity from the known neutral amino acid
carriers(5, 6) . Uptake of GSH was saturable and was
inhibited by organic anions consistent with a carrier-mediated process.
Brain perfusion studies further showed transendothelial transport of
GSH into the brain and partial inhibition of uptake into endothelium by
addition of BSP-GSH (7) and removal of sodium from the
perfusion medium. (
)If GSH uptake is to have physiologic
significance and therapeutic potential, a driving force is required to
overcome the steep electrochemical gradient from plasma to endothelial
cell cytoplasm. The previous results support the possible existence of
a luminal Na
-dependent GSH transporter for uptake and
abluminal transporter for efflux of GSH in brain endothelium. However,
in other systems, such as kidney, as well as in brain, it has been
argued that GSH uptake does not exist and that nearly all apparent GSH
uptake depends on the breakdown of GSH initiated by GGT(8) .
Since GGT and GSH transporter(s) are functional at the BBB, we
decided to see if these activities could be resolved by size
fractionation and oocyte expression and if a
Na-dependent GSH uptake could be demonstrated
unequivocally. We have recently cloned two liver transporters and
demonstrated that the sodium-independent, BSP-GSH insensitive rat
canalicular GSH transporter (RcGshT), but not rat sinusoidal GSH
transporter (RsGshT), was present in brain(9, 10) .
Therefore, an additional goal was to determine if a putative
sodium-dependent GSH transporter could be separated from RcGshT.
S-Labeled isotopes (glutathione, 145 Ci/mmol;
cysteine, 600 Ci/mmol) and
[glycine-2-
H]glutathione (1000 Ci/mmol)
were obtained from DuPont NEN. Chemicals and buffer reagents were
obtained from Sigma. BSP-GSH was synthesized in the laboratory as
described previously(9) .
Aliquots (1 µl) of the
plasmid pool (20 µl) prepared from each fraction's cDNA
library were used to transform Escherichia coli DH 5a strain.
Plasmid DNA was purified from fraction 3-15 transformants and
linearized with NotI and transcribed in vitro with T7
RNA polymerase in the presence of GpppG cap using a protocol supplied
with riboprobe transcription system (Promega). The complementary RNAs
(cRNAs) were injected into Xenopus oocytes (3 ng/oocyte) which
were then analyzed for expression of GSH transport 3 days after
injection. Water-injected oocytes served as controls. The activity of
individual fractions for GSH uptake was compared with that of total
mRNA-injected oocytes under GGT-inhibited (1 mM acivicin, 30
min) or uninhibited conditions. GSH uptake in active fractions was
determined in the presence of Na (NaCl medium) or
absence of Na
(sucrose medium or choline chloride
medium). In some experiments, effect of BSP-GSH (2 mM) on GSH
uptake in fraction 7 cRNA-injected oocytes was determined in NaCl
medium at 0.05 mM and 2 mM GSH concentrations. The
molecular form of uptake in acivicin-pretreated or untreated fraction 7
cRNA-injected oocytes was determined by HPLC (13) in
double-labeled experiments in an incubation medium containing
[
S]GSH (1 µCi) +
[
H]GSH (2.5 µCi) + 0.1 mM unlabeled GSH.
Kinetics of GSH uptake in the
Na-dependent fraction was carried out by adding
varying concentrations of unlabeled GSH (0.01, 0.05, 0.1, 0.2, 0.5, 1,
2, 5, 10, 20, and 50 mM containing 10 mM DTT) to the
NaCl incubation medium containing 4-8 ng of cRNA-injected oocytes
and measuring uptake after 1 h(12) . Pilot experiments on GSH
uptake at two extreme concentrations of GSH (1 mM and 50
mM) for 15, 30, 60, and 120 min established that the uptake
was linear during the incubation period studied, and a 1 h time point
from the linear part of the curve was chosen for kinetic studies.
Figure 1:
Uptake of GSH into
bovine brain capillary mRNA-injected and guinea pig capillary
mRNA-injected oocytes. Oocytes were injected with either water or mRNA
(30 ng) and maintained at room temperature for 3 days. Injected oocytes
were then washed and incubated in either Na-containing
(NaCl) or Na
-free (sucrose) medium in the presence of
[
S]GSH (5 µCi in 10 mM DTT plus 10
mM GSH) for 1 h. Uptake is expressed as nanomoles/oocyte/h.
Both bovine and guinea pig mRNA-injected oocytes showed a significant (p < 0.05, indicated by asterisk) decrease in
uptake in sucrose medium (hatched bars) as compared to NaCl
medium (open bars). Water-injected oocytes showed similar, low
uptake in both media (solid bar).
Figure 2:
GSH transport activity in mRNA size
fractions isolated by size fractionation of cDNA. The complementary
RNAs from each fraction (3 ng/oocyte) were injected into two different
oocyte preparations. After 3 days, oocytes were pretreated with
acivicin and were incubated in NaCl medium containing 10 mM GSH. Individual data from two oocyte preparations (points) and their mean (bars) are presented for each
size fraction. On the upper left side, GSH uptake in oocytes
after injection of bovine capillary total poly(A) RNA
(30 ng) and water-injected oocytes (marked C) are shown. Inset shows the presence of the 4.0-kb RcGshT transcript by
Northern blot analysis in only one of the size fractions (fraction 5)
that showed GSH transport activity. On the upper right side,
Northern blot analysis demonstrated absence of GGT transcript in
fraction 7 while fraction 11 contained GGT. 1 µg of cRNA size
fractions were electrophoresed as above for RcGshT except that
hybridization was carried out with a
P-labeled 2.0-kb
insert of human GGT cDNA. Lane B shows the presence of GGT
transcript in bovine brain used as positive
control.
Figure 3:
Effect of replacement of Na with choline chloride on GSH uptake at 0.05 mM GSH (A) or 2 mM GSH (B) in non-RcGshT-containing
active size fractions 7 and 11. GSH uptake was studied in oocytes that
were pretreated with acivicin to inhibit GGT. A significant decrease in
net uptake (after subtracting uptake in water-injected controls) was
observed when Na
was removed from the incubation
medium of oocytes expressing fraction 7, while Na
removal did not affect uptake in fraction 11. Data are mean
± S.E. from 3 separate experiments on different oocyte
preparations each performed in duplicate.
BSP-GSH at 2 mM concentration caused
a significant inhibition of GSH uptake in fraction 7 cRNA-injected
oocytes. Thus, in 1-h incubation experiments in NaCl medium with 0.05
mM and 2 mM GSH, BSP-GSH inhibited GSH uptake by
90% and 39%, respectively.
In addition to demonstrating the
absence of GGT in fraction 7 (see above, Fig. 2), supporting
evidence for the dissociation of fraction 7 from GGT-mediated
degradation and resynthesis was obtained in additional studies. Net
uptake of GSH in fraction 7 cRNA-injected oocytes was similar under
GGT-inhibited or uninhibited conditions (for example, uptake
measurements in the presence or absence of acivicin at 0.05 mM GSH (0.17 ± 0.02 and 0.15 ± 0.03 nmol/oocyte/h,
respectively) and at 2 mM GSH (1.23 ± 0.11 and 1.16
± 0.20 nmol/oocyte/h, respectively) were not significantly
different from each other). The molecular form of uptake in S- and
H-double-labeled experiments in
fraction 7 cRNA-injected oocytes was predominantly intact GSH with both
isotopes in the presence or absence of acivicin (Fig. 4), and
the ratios of the
S/
H in GSH peak in cells in
the presence or absence of acivicin corrected for spillover (1.112 and
1.104, respectively) were also very similar to that of the incubation
medium (1.091). Furthermore, HPLC analysis of uptake of
[
S]cysteine (+50 µM unlabeled
cysteine in 10 mM DTT) showed that nearly all the
radioactivity was associated with cysteine and no conversion to GSH had
taken place in the 1-h incubation period (not shown). Thus, breakdown
and resynthesis could not account for apparent GSH uptake.
Figure 4:
HPLC chromatogram showing molecular form
of uptake of tracer [H]GSH and
[
S]GSH (in the presence of 0.1 mM unlabeled GSH) in oocytes injected with cRNA from fraction 7.
Oocytes were either pretreated with acivicin (A) or untreated (B) and uptake was performed in NaCl medium for 1 h. HPLC was
performed in a Shimadzu HPLC system equipped with a UV detector
connected to a Beta RAM Flow-Through Monitor (IN/US Systems Inc.,
Fairfield, NJ) as described previously(6, 7) . The
predominant radioactivity peak for both isotopes was GSH, and the
S/
H ratios in the GSH peak under GGT-inhibited
and noninhibited conditions were nearly identical with the ratio in the
GSH peak in the uptake medium (C). The arrows identify the elution peak for the authentic unlabeled GSH in each
chromatogram.
Fig. 5shows the kinetics of uptake of GSH in oocytes injected
with cRNA from fraction 7 versus water controls. The uptake
was determined in NaCl medium in oocytes that were preincubated with
acivicin. SAAM analysis of the fits to the data (5, 6) showed two components, a high affinity
Michaelis-Menten component with a K of 0.40
± 0.19 mM and a sigmoid low affinity component with a K
of 10.8 ± 3.1 mM and a Hill
coefficient of n = 2. In view of a predominant
sodium-dependent component (Fig. 3), kinetics were not performed
in Na
-free medium to establish if the small
Na
-independent component in fraction 7 is saturable.
This analysis has been postponed until completion of cloning so that
more meaningful information can be obtained on the kinetics of the
sodium-independent component of the isolated Na
-GSH
cotransporter.
Figure 5:
Concentration-dependent uptake of GSH in
oocytes expressing bovine capillary size fraction 7 and water controls.
Oocytes were injected with water or cRNA, and uptake was carried out 3
days later. The incubation medium (NaCl medium) contained 2 µCi of
labeled GSH and varying concentrations of unlabeled GSH, and uptake was
studied for 1 h. The data in the open circles represent net
uptake after subtraction of the water controls, shown as closed
circles. The net data points (mean ± S.E., n = 3) were fitted with SAAM using the Michaelis-Menten
equation which showed a high affinity component (inset) with a K of 0.40 ± 0.19 mM (V
= 0.57 ± 0.21
nmol/oocyte/h) and a sigmoid low affinity component with a K
of 10.8 ± 3.1 mM (V
= 7.66 ± 0.27
nmol/oocyte/h) and a Hill coefficient of n =
2.
Uptake of GSH in oocytes expressing brain capillary mRNA has
been demonstrated in this study for the first time. Three distinct GSH
transporter activities have been identified based on size
fractionation, one of which corresponds to the previously cloned sodium
independent rat canalicular GSH transporter. One of the remaining two
GSH transporter activities was found to be
Na-dependent and exhibited both high affinity and low
affinity GSH transport. This also is the first unequivocal
demonstration of a sodium-dependent transporter by expressing its mRNA
in a heterologous cell which normally does not express it and therefore
provides the strongest evidence to date that sodium-dependent GSH
transporter exists.
Because of the widely held view that GSH is
broken down by ecto GGT and resynthesized to GSH within the
cell(14) , we have ensured that GGT (and dipeptidase) mediated
breakdown, constituent amino acid uptake and GSH resynthesis were
negligible, and uptake of labeled GSH in brain capillary mRNA and size
fraction cRNA-injected oocytes was due to its transport in intact form.
This was achieved by several approaches. (a) GSH transport
studies were carried out under conditions of GGT inhibition by
acivicin. (b) Molecular forms of uptake in capillary mRNA or
cRNA-injected oocytes were determined by HPLC to be predominantly GSH
with or without GGT inhibition. (c) In uptake experiments with
oocytes expressing the novel sodium-dependent GSH transporter,
double-labeled GSH was employed to exclude breakdown and resynthesis:
the isotope ratio of GSH taken up was nearly identical with that of the
medium in the presence or absence of acivicin. (d) Transport
of [S]cysteine examined under conditions of GSH
uptake (1-h incubation, 18 °C, NaCl medium, GGT inhibition) showed
that radioactivity taken up was predominantly cysteine, and no
conversion to GSH was evident by HPLC. (e) Finally, it was
unequivocally shown that the fraction expressing
Na
-dependent GSH transporter did not contain a
transcript for GGT in Northern blot analysis when probed with a
full-length GGT clone. The existence of Na
/GSH
cotransport has been suggested from physiological studies in cells or
membrane vesicles of the kidney, intestine, and lung (15, 16, 17) as well as our own brain
perfusion studies(7) . However, controversy has surrounded the
validity of these observations, others suggesting that even a small
fraction of GGT escaping inhibition or impurities in radiolabel could
account for the findings as hydrolysis, sodium amino acid co-transport,
and intracellular GSH resynthesis(18) . Thus, our expression in
oocytes of a Na
-dependent GSH transport which can be
dissociated from GGT unequivocally supports the existence of such a
system.
The exact localization of the GSH transporters is not clear
at the present time, but some speculations can be made. RcGshT, one of
the transporters identified in the brain, is a
Na-independent, low affinity, BSP-GSH insensitive GSH
transporter. Since its function is as an effluxer under physiological
conditions, its presence in the whole brain and brain capillaries would
suggest that it is probably different from the luminal brain
endothelial GSH transporter, which was shown to be
Na
-dependent and BSP-GSH sensitive in our in situ brain perfusion studies(7) . RcGshT may function to efflux
GSH from the endothelial cells of the BBB to the parenchyma. Since
astrocytes are known to efflux GSH at a high rate (19) , RcGshT
may also serve to efflux astrocyte GSH. Support for this view was
obtained in our studies confirming GSH transport in cultured neonatal
rat astrocytes and demonstrating the presence of RcGshT transcript by
Northern blot analysis.
GSH transport at the BBB can be
envisioned to proceed by endothelial uptake at the luminal pole that is
Na-dependent. This system has the possibility of being
concentrative so that low plasma GSH (10-20 µM) can
lead to net accumulation of GSH in endothelial cells, due to the likely
coupling to the entry of sodium. At the same time, the
GGT-
-glutamyl cycle could provide another mechanism for handling
luminal GSH and maintaining brain GSH homeostasis. The relative
importance of these two mechanisms is not known and may depend on
species, age, or other factors. Furthermore, redundancy of mechanisms
for maintenance of such a vital defense factor in brain would not be
surprising.
The apparent GSH transport activity expressed by
fraction 11 will need further characterization. Since GGT is present in
this fraction, the possibility of breakdown and resynthesis will need
to be rigorously evaluated. The molecular form taken up in the presence
of acivicin was intact GSH, and cysteine was not incorporated into GSH
in oocytes expressing fraction 11. No hybridization signal
was observed when this fraction was probed with RsGshT and RcGshT,
which suggests that this is not another family member of either and
also that alternate processing is unlikely. Thus, a novel, previously
undescribed sodium-independent GSH transporter is suggested.
In summary, we have demonstrated for the first time that mRNA of GSH transporters other than the two we previously have cloned from rat liver exist, indicating that there are multiple GSH transporters which may have cell-specific expression and may govern uptake or efflux of GSH. Also, we have shown for the first time that brain capillaries express a sodium-dependent GSH transporter and that its apparent activity cannot be explained by the commonly held view of the combination of breakdown of GSH, sodium-dependent amino acid uptake, and GSH resynthesis; this provides the strongest evidence heretofore available of the occurrence of a sodium-dependent GSH transporter. Furthermore, the presence of sodium gradient-driven uptake of GSH provides a physiologic driving force for net GSH transport against the electrochemical gradient.