-Alanine and
-fluoro-
-alanine concentrative transport
in rat hepatocytes is mediated by GABA transporter GAT-2
Mengping
Liu1,
Rosalind L.
Russell1,
Leonid
Beigelman2,
Robert E.
Handschumacher1, and
Giuseppe
Pizzorno1
1 Departments of Internal
Medicine (Oncology) and Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06520; and
2 Ribozyme Pharmaceuticals,
Boulder, Colorado 80301
 |
ABSTRACT |
Studies on the compartmentalization of uridine
catabolic metabolism in liver have indicated accumulation of
-alanine as well as
-fluoro-
-alanine (F
AL) for
5-fluorouracil in the hepatocytes. Using preparations of rat
hepatocytes we were able to identify a
Na+-dependent transport with high
affinity for
-alanine and GABA with Michaelis constant
(Km) of 35.3 and 22.5 µM, respectively. A second
Na+-dependent kinetic component
with Km >1 mM
was also identified. The sigmoidal profile of
-alanine uptake with
respect to Na+ shows the
involvement of multiple ions of sodium in the transport process. A Hill
coefficient of 2.6 ± 0.4 indicates that at least two sodium ions
are cotransported with
-alanine. The flux of
-alanine was also
shown to be chlorine dependent. The substitution of this anion with
gluconate, even in the presence of
Na+, reduced the intracellular
concentrative accumulation of
-alanine to passive diffusion level,
indicating that both Na+ and
Cl
are essential for the
activity of this transporter. The transport of
-alanine was
inhibited by GABA, hypotaurine,
-aminoisobutyric acid, and F
AL in
a competitive manner. However, concentrations up to 1 mM of
L- and
D-alanine, taurine, and
-aminoisobutyric acid did not affect
-alanine uptake. Considering
the similarities in substrate specificity with the rat GAT-2
transporter, extracts of hepatocytes were probed with the anti-GABA
transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was
present in the hepatocyte and in the GAT-2 transfected Madin-Darby
canine kidney cell extract, confirming the extraneural localization of
this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be
explained with the formation of
-alanine and F
AL and their effect
on the cellular reuptake of GABA.
uridine; liver; 5-fluorouracil
 |
INTRODUCTION |
-ALANINE AND
-fluoro-
-alanine (F
AL) are the major products of uracil and
5-fluorouracil catabolism. This process occurs primarily in the liver
and is specifically localized in parenchymal cells (15). In earlier
studies by Diasio and co-workers (25) it was shown that high
concentrations of F
AL accumulated initially in kidney and liver, and
later on it was found in several other tissues, including eyes and
small intestine. These data suggested that an active concentrative
process may be responsible. In unrelated studies by Miyamoto et al.
(16) and Turner (23), the presence of an active
Na+-dependent concentrative
mechanism for
-alanine and
-amino acids was demonstrated in
brush-border vesicles from small intestine and proximal renal tubules
(23). Other studies in astrocytes and neurons also established the
ability of these cells to concentrate
-alanine by a
Na+-dependent process (9,
14).
Our interest arises from studies on the localization of uridine
degradation products in the liver (15). We confirmed that
-[3H]alanine and
[3H]F
AL accumulated
in the hepatocytes following incubation with [6-3H]uracil and
[5,6-3H]fluorouracil.
We also demonstrated these
-amino acids were concentrated by a
Na+-dependent process. Others have
characterized the transport of taurine, another
-amino acid, in rat
hepatocytes, showing a single Na+-dependent transport system at
low concentration of taurine (5, 8). The transport of taurine was shown
to be competitively inhibited by
-alanine and hypotaurine (8). Two
distinct Na+-dependent
-amino
acid transport systems, one with high affinity and the other with low
affinity and high capacity, have been characterized in two renal
epithelial cell lines, LLC-PK and Madin-Darby canine kidney (MDCK), and
in the proximal tubule cells from rabbit and human origin (10-12).
In this study we characterize the
Na+- and
Cl
-dependent
-alanine
and F
AL transporter and provide evidence that it is identical to the
GABA transporter designated GAT-2 on the basis of kinetic, substrate,
immunological, and inhibition studies.
 |
MATERIALS AND METHODS |
Chemicals.
-[3-3H]alanine (87 Ci/mmol) and
[3H]H2O
(18 Ci/mmol) were purchased from NEN (Boston, MA).
[2,3-3H]GABA (91 Ci/mmol) and
[carboxy-14C]inulin
(10 mCi/mmol) were purchased from Amersham (Arlington Heights, IL).
Taurine, hypotaurine, L- and
D-alanine,
-aminoisobutyric acid, and
-aminoisobutyric acid were obtained from Sigma Chemical (St. Louis, MO). (R,S)-F
AL was purchased from American Tokyo Kasei
(Portland, OR). All other reagents were of the highest grade available.
Hepatocyte preparation.
Rat hepatocytes, provided by the generous assistance of the Yale Liver
Center, were isolated by the collagenase perfusion technique (2, 21).
Male Sprague-Dawley rats (150-200 g) from Charles River
Laboratories were anesthetized with 50 mg/kg pentobarbital by
intraperitoneal injection. Heparin (500 U/100 g body wt) was injected
into the abdominal vena cava, and the livers were perfused through the
portal vein with calcium-free perfusion buffer (pH 7.4, 37°C)
saturated with a gas mixture of 95% oxygen and 5% carbon dioxide at
38 ml/min for 10 min. Extending the preperfusion period beyond this
time period reduced the metabolic and transport activity of both cell
populations. The calcium-free perfusate was changed to buffer
containing 0.025-0.05% collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.5 mM
CaCl2. Perfusion was continued for
10 min at 38 ml/min. The liver was removed from the animal and combed
into 50 ml of pH 7.4 L-15 medium (GIBCO, Grand Island, NY) saturated
with O2. The cell suspension was
filtered through 45-µm mesh nylon fabrics (Tetko), and the filtrate
was sedimented at 50 g. The hepatocyte
pellet was washed three times with cold L-15 medium and resuspended at
4-6 × 106 cells/ml in
L-15 medium. Hepatocyte preparations obtained by Percoll centrifugation
(21) were >95% pure, and the cell viability, determined by trypan
blue exclusion, was in excess of 85% before use and at least 80% at
the end of uptake experiments.
Transport studies.
Hepatocyte suspensions were centrifuged at 750 g for 5 min in a Sorvall GLC-4
centrifuge. The pellet was washed twice with 150 mM
Na+ or 150 mM choline
(Na+ free) Hanks' balanced salt
medium containing 5.5 mM
D-glucose and 4 mM HEPES buffer
(pH 7.4). In Cl
-free
transport experiments, NaCl and KCl were replaced by their corresponding gluconate salts. The pellet was resuspended in the appropriate medium to give a final cell density of 4-6 × 106 cells/ml.
The oil-stop method for nucleoside transport studies previously
reported (6) was used to assess transport. Uptake of
-[3H]alanine and
GABA was initiated by mixing 30 µl of cell suspension with 60 µl of
radioactive substrate in Hanks' medium in a 1.5 ml Eppendorf microfuge
tube. At appropriate time intervals, 60 µl of the mixture were placed
in a 400-µl Eppendorf microfuge tube containing 125 µl of oil (16%
Fischer 0121 light paraffin oil and 84% Dow Corning 550 silicon fluid;
final specific gravity 1.04 g/ml) layered over 30 µl of 15%
trichloroacetic acid and centrifuged in a Beckman model B microfuge for
30 s at 10,000 g. The tubes were cut
through the oil layer, and radioactivity in each half was assayed in 5 ml of Ecolite(+) after vigorous vortexing. "Time
zero" values for uptake, attributed to the
extracellular radioactivity trapped in the cell pellet, were determined
by spinning 20 µl of hepatocytes through a layer of radioactive
substrate (40 µl) placed over the oil in the oil-stop tube. The
intracellular volume of hepatocytes was calculated (6) in all
experiments by using
[3H]H2O
to measure the total water space and
[14C]inulin for
extracellular space. Radioactivity was determined in a Beckman LS 7000 scintillation counter.
In all the kinetic studies an uptake interval of 2-5 min uptake of
-[3H]alanine and
GABA was utilized; however, a 20-min interval was selected for all
competition experiments. Paper chromatography with butanol-acetic
acid-H2O (25:4:10) was used to
demonstrate that radiolabeled alanine and GABA remained intact after
exposure to hepatocytes. More than 90% of radioactivity from
-alanine or GABA incubated in hepatocyte suspensions for 20 min at
room temperature migrated to an area identical to that for
-alanine or GABA.
Uptake determinations were routinely done in triplicate and repeated
with two or three different hepatocyte preparations on different days.
The results are presented as the means ± SE. Student's t-test for unpaired samples was used
to test the significance of difference among means
(P < 0.05 was considered
significant). Kinetic data were analyzed using the Enzfitter program
(R. J. Leatherbarrow, published by Elsevier-Biosoft) for nonlinear
least-squares fit of the Michaelis-Menten equation.
Western blot analysis.
Rat hepatocytes and MDCK cells, transfected with GAT-2 cDNA (provided
by Dr. Michael J. Caplan, Yale University School of Medicine, New
Haven, CT), were solubilized in 2× SDS gel loading buffer (100 mM
Tris · HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and heated to 100°C for 10 min. The lysate was separated on a discontinuous 7.5%
SDS-polyacrylamide electrophoresis system (7, 13, 19). Separated
proteins were transferred to polyvinylidene difluoride membranes
(Hybond P, Amersham) and blocked with a 5% casein buffer (5% casein,
0.05% Triton X-100, 0.3 M NaCl, 50 mM citric acid, 0.3 M Tris base, pH
7.6). The membranes were incubated overnight at 4°C with polyclonal anti-GABA transporter antibody (R-22) diluted 1:100 in casein buffer
(1). The membranes were washed three times (5 min each) with PBS buffer
containing 0.05% Tween 20 (PBST) and incubated with 1:2,000 diluted
secondary antibody (donkey anti-rabbit horseradish peroxidase
conjugated antibody; Amersham) for 1 h at room temperature. The
membranes were then washed with PBST (3 × 5 min) and detected with enhanced chemiluminescence reagents (Amersham).
 |
RESULTS |
Studies of the homeostasis and metabolic fate of uridine in the liver
(15) indicated that
-alanine, the primary breakdown product of
uracil, remains in the hepatocytes in high concentrations when they are
exposed to uracil, a process dependent on sodium (Fig.
1). Biphasic kinetics (Fig.
2) are observed with a high affinity
concentrative component [Michaelis constant
(Km) 35.3 ± 4.4 µM and a maximal velocity
(Vmax)
22.7 ± 2.5 µM/min]. The apparent
Km of the
concentrative transporter is significantly greater than circulating
levels of
-alanine and thus may be determinant under physiological
conditions. A second Na+-dependent
kinetic component is also present with an apparent Km of at least 2 mM. The zero transport time course of accumulation of
-alanine
within the cells is also consistent with a very rapid passive diffusion
component that leads to equilibration with the medium followed by
active concentration, which only occurs in the presence of sodium. When
sodium is replaced by either lithium or choline,
-alanine still
rapidly equilibrates with the intracellular water but is not
concentrated (Fig. 1). In the presence of
Na+, concentrative transport of
-alanine, after an initial burst, remains linear for >20 min, and
>90% of the radioactivity remained associated with
-alanine.

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Fig. 1.
Time course of
-[3H]alanine uptake
by rat hepatocytes. Cells were suspended in either
Na+-containing medium ( ) or
Na+-free medium
[Li+ replacement ( ) and
choline replacement (+)]. Final concentration of -alanine
during incubation was 25 µM. After appropriate time intervals uptake
was terminated by the oil-stop method as described in
MATERIALS AND METHODS. Intracellular
concentrations were based on intracellular water volume. Values are
means ± SE of triplicate estimates.
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Fig. 2.
Substrate dependence of total and
Na+-dependent -alanine
transport in rat hepatocytes. Hepatocytes were incubated in
Na+-containing medium and in
Na+-free medium with varying
substrate concentrations. Transport was measured as described in
MATERIALS AND METHODS. The
Na+-dependent component of
transport was obtained by subtracting, at each substrate concentration,
the Na+-free (choline replacement)
part from total (inset represents Eadie-Hofstee plot of -alanine
uptake by hepatocytes at substrate concentrations up to 2 mM).
Michaelis constant values for the high affinity process were determined
as described.
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|
To determine the stoichiometric relationship between sodium ion entry
and the transport of uridine, the dependence of the rate of transport
on sodium concentration was determined. A sigmoidal response was
obtained (Fig. 3) which is consistent with
a cooperative mechanism that requires at least two and probably three
sodium ions for each mole of
-alanine (Hill coefficient 2.6 ± 0.4). These results suggest a mechanism in which the binding of the first sodium ion increases the affinity of the carrier for the second
sodium ion and the participation of a single sodium ion is insufficient
to permit the active concentration of
-alanine.

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Fig. 3.
Effect of external Na+
concentrations on Na+-dependent
-[3H]alanine
transport. -alanine (25 µM) uptake in hepatocyte was determined in
presence of varying Na+
concentrations. Isosmolarity was maintained by replacement of
Na+ with choline. Each point is
mean of triplicate determinations. Inset presents linear transformation
of data as expressed by the Hill equation.
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|
This Na+-dependent transport
system was also Cl
dependent. Substituting this anion with gluconate in the transport
buffer reduced the rate of uptake and accumulation of
-alanine to
passive diffusion level (Fig. 4).
Increasing concentrations of
Cl
resulted in a
concentrative uptake of
-alanine that exhibited a hyperbolic
dependence, suggesting that one
Cl
is associated with the
transport of one molecule of
-alanine (data not shown).

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Fig. 4.
Time course of
-[3H]alanine uptake
by rat hepatocytes in presence and absence of
Cl . Cells were suspended in
either Cl -containing medium
or Cl -free medium
(gluconate replacement). Final concentration of -alanine during
incubation was 25 µM. After appropriate time intervals uptake was
terminated by oil-stop method as described in
MATERIALS AND METHODS. Intracellular
concentrations were based on intracellular water volume. Values are
means of 2 experiments conducted in triplicate.
|
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The Na+- and
Cl
-dependent transporter is
highly substrate specific. L- or
D-alanine, taurine, and
-aminoisobutyric acid did not inhibit
-alanine accumulations at
concentrations up to 1 mM (Fig. 5).
However, GABA and hypotaurine, the sulfonic acid analog of
-alanine,
were potent inhibitors of this process (Fig. 5 and Table
1). F
AL, the primary catabolic product
of 5-fluorouracil, also exhibits inhibitory properties on this
transport system. All three compounds display competitive inhibition
kinetics (data not shown).

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Fig. 5.
Substrate specificity of -alanine transport by rat hepatocytes.
Hepatocytes were incubated in
Na+-containing medium with
-[3H]alanine (25 µM) for 20 min in absence or presence of -alanine structural
analogs using range of inhibitor concentrations (0-1 mM). Relative
velocities are expressed as percentage of rate of -alanine uptake
determined in absence of -alanine analogs and represent the mean of
triplicate determinations. -AIBA, -aminoisobutyric acid, F AL,
-fluoro- -alanine; HT, hypotaurine.
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|
Earlier studies using PCR (4) have demonstrated the existence of a
specific GABA transporter, termed GAT-2, present in liver, kidney, as
well as in brain. Because of its possible identity with the
-alanine
transporter, we have characterized the transport of GABA in hepatocytes
and demonstrated an absolute dependence of the concentrative phase on
the presence of Na+. A minor
degree of concentration may have occurred when
Na+ is replaced by
Li+ but not choline (Fig.
6). It is apparent in this system as well as with
-alanine that GABA rapidly enters cells by a
Na+-independent system to
approximately equilibrate with the medium. As with
-alanine, the
sodium-dependent uptake exhibits a high affinity for GABA (22.5 ± 2.8 µM) and a somewhat lower
Vmax (18.5 µM/min) (Fig. 6 and Table 1).

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Fig. 6.
Time course of
[3H]GABA uptake by rat
hepatocytes. Cells were suspended in l50 mM NaCl, l50 mM LiCl, or l50
mM choline chloride buffer and final concentration of GABA was 25 µM
during uptake measurements. After appropriate time intervals uptake was
terminated by oil-stop method as previously described. Values are means ± SE of triplicate determinations.
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|
To establish the identity between the
-alanine
Na+- and
Cl
-dependent transporter in
rat hepatocytes and the GAT-2 transporter, we probed protein extracts
from hepatocytes with an anti-GABA transporter antibody R-22 (1). As
shown in Fig. 7, an 80-kDa protein
corresponding to the GAT-2 transporter was present in the hepatocytes
extract and in the lysate from MDCK cells expressing GAT-2, used as
positive control.

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Fig. 7.
Western blot analysis of GAT-2 antigen in rat hepatocytes and
nonparenchymal cells. Rat hepatocyte and MDCK cell proteins were
separated on a 7.5% SDS-PAGE gel and analyzed by Western blot as
described in MATERIALS AND METHODS.
Lanes 1-3, hepatocyte lysates
containing 100, 100, and 50 µg of total protein, respectively;
lanes 4 and
5, extracts of MCDK cells transfected
with GAT-2 cDNA.
|
|
 |
DISCUSSION |
The compartmentalization of uridine catabolism in liver resulting in
the specific accumulation of
-alanine and F
AL, in the case of
5-fluorouracil, led to the characterization of this
Na+- and
Cl
-dependent transporter
for
-amino acids and GABA, a system that had poor affinity for
taurine. This transporter requires the presence of both specific cation
and anion. Without one or the other no significant active transport of
-alanine and GABA is observed. Thus to demonstrate the cation
dependency of this transporter as in Fig. 1, the chloride ion must be
present; similarly, as shown in Fig. 4, to prove anion dependency, the
transport buffer must contain sodium ion.
The Hill coefficient of 2.6 ± 0.4 for
Na+ and one
Cl
for
-alanine is
similar to other
-alanine and
-amino acid transport systems
previously characterized in rat hepatocytes (5, 8), renal brush-border
membranes (23), kidney cells derived from the proximal tubules (10,
11), and intestinal brush-border membranes (16). In all these transport
systems, however, taurine was also transported and was able to
efficiently inhibit the
-alanine transporter in these tissues. In
rat hepatocytes taurine had only a very limited inhibitory effect on
-alanine uptake (~40%) even at 1 mM.
Kinetic characteristics, substrate specificity, and antibody
recognition all suggest that the
Na+- and
Cl
-dependent transporter
here described is the GAT-2 transporter. Unlike other GABA transporters
previously identified, studies from Borden et al. (4) have indicated
that GAT-2 is present not only in the central nervous system but also
in other nonneural tissues such as liver, kidney, and retina.
At the neural level GAT-2 is localized in the leptomeninges surrounding
the brain, possibly suggesting its role in regulating GABA levels in
the cerebrospinal fluid, therefore affecting GABAergic transmission
indirectly, or playing a role in the osmoregulation. GAT-2 is absent in
neuronal cultures or in neurons in vivo. The basolateral distribution
of GAT-2 in MDCK cells (1) and its presence in the leptomeninges also
suggest that this transporter is involved in the clearing of GABA from
the cerebrospinal fluid in nonsynaptic regions (3).
Neurological toxicity has been reported following administration of
5-fluorouracil (17, 20), particularly for regimens using 24-h infusion
of high-dose 5-fluorouracil (2,600 mg/m2) where approximately 6%
of the patients developed treatment-related encephalopathy (24). The
clinical manifestations include disorientation, confusion, agitation,
seizure, stupor, and coma (24). The affinity of
-alanine and F
AL
for the GAT-2 transporter raises the possibility that the neurotoxicity
seen with 5-fluorouracil therapy may relate to an effect of F
AL on
the reuptake of GABA, an inhibitory neurotransmitter. Administration of
F
AL has been shown in cats and dogs to induce a drowsy and stuporous
state and struggling that are similar to those produced by oral
administration of 5-fluorouracil (18). Histologically two main changes
were described: 1) vacuole formation and 2) necrotic lesions. The
distribution of these effects was not diffuse but limited to the
cerebellar nuclei and white matter, tectum, and tegumentum of the brain
stem. The selective localization of the neuropathological changes may
be due to the preferential accumulation of F
AL and an inhibitory
effect on the function of the GAT-2 transporter.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Chee-Wee Lee (Dept. of Physiology, National University
of Singapore, Singapore) for the helpful discussion in the preparation
of this manuscript.
 |
FOOTNOTES |
The Madin-Darby canine kidney cells transfected with GAT-2 cDNA and the
R-22 anti-GABA transporter antibody were generously provided by Dr.
Michael J. Caplan (Dept. of Cellular and Molecular Physiology, Yale
University School of Medicine, New Haven, CT). The hepatic cells were
kindly supplied by the Liver Research Core Center, which was supported
in part by the National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-34989 and the National Institutes of Health Grant R25
(CA-47883-03).
This work was supported by grants from the American Cancer Society
(CH67) and National Cancer Institute (CA-08341, CA-45303, and
CA-67035).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. Pizzorno, Dept. of Internal Medicine
(Oncology), Yale Univ. School of Medicine, New Haven, CT 06520.
Received 8 April 1998; accepted in final form 15 September 1998.
 |
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