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INTRODUCTION |
Free amino acids need to be transported from the lumen of
the intestine as well as from the urinary filtrate of kidney tubules into the extracellular space. This transcellular (re)absorption involves the passage across both the luminal and the basolateral membrane of epithelial cells. Most neutral amino acids are taken up
through the luminal membrane of these cells by a
Na+-dependent electrogenic transport system
that was named B0 or B for kidney and intestine,
respectively, and that is not molecularly defined as yet (1, 2).
The import of cationic amino acids and of L-cystine depends
on the expression of a heteromeric transporter that is composed of a
multitransmembrane span catalytic subunit (glycoprotein-associated amino acid transporter, light chain) named
b0,+AT1 (broad
specificity, Na+-independent neutral and cationic amino
acid transporter) and the covalently associated type II glycoprotein
(heavy chain) rBAT (related to b0,+ amino acid transport)
(3-6). Genetic studies have shown that defects in the genes encoding
either subunit lead to cystinuria (4, 7, 8). The function of this
transporter has been characterized in Xenopus oocytes first
by investigating the function of exogenous rBAT associated with an
endogenous b0,+AT and then, upon identification of the
mammalian b0,+AT subunit, by measuring the function of the
mammalian heterodimer expressed as fusion protein in Xenopus
oocytes or in COS-7 cells (3, 5, 6, 9-11). These studies indicated
that b0,+AT-rBAT functions as an obligatory exchanger and
suggested that its major mode of transport is, at a normal membrane
potential, the uptake of extracellular cationic amino acids or
L-cystine that are exchanged against intracellular neutral
amino acids. These neutral amino acids could in turn be recycled into
the cell by system B/B0. Recently, we have characterized
the biosynthesis, localization and uptake function of
b0,+AT-rBAT expressed in MDCK cells (12). This study has
shown in an epithelial context that the surface expression of
b0,+AT and rBAT depends on their association, which is
necessary for the maturation and stabilization of rBAT. This study has
also confirmed that b0,+AT-rBAT displays extracellularly a
high apparent affinity for L-cystine > cationic amino
acids (L-Arg) > large neutral amino acids
(L-Leu).
The basolateral efflux of amino acids is as yet less well understood
than the apical influx. The two heteromeric amino acid transporters,
composed of the glycoprotein subunit (heavy chain) 4F2hc
(CD98) and an associated catalytic subunit, y+LAT1 or LAT2,
that are highly expressed in the proximal kidney tubule and the small
intestine, were shown in Xenopus oocytes to function as
obligatory exchangers (13-17). The physiological function of
y+LAT1-4F2hc appears to be the electroneutral
efflux of intracellular cationic amino acids that are exchanged for
extracellular large neutral amino acids together with Na+.
This mode of transport is based on the results of expression experiments made in Xenopus oocytes and is compatible with
the phenotype of patients carrying the genetic disease lysinuric
protein intolerance that was shown to be due to a defect in the
corresponding gene (13, 14, 18, 19). As this system needs to function in the context of the (re)absorption of all amino acids, we postulate that an additional export system that recycles the imported neutral amino acids needs to be expressed in the basolateral membrane.
The function of the second heteromeric exchanger,
LAT2-4F2hc, has been extensively studied in
Xenopus oocytes (15-17, 20). This transporter
preferentially exchanges middle-sized and large neutral amino acids
across the membrane. Importantly, its apparent affinities for various
amino acids is much lower inside compared with outside of the cells
(with an exception for Gly), suggesting that its activity depends on
the intracellular amino acid availability (15). It was suggested that
its physiological role is to equilibrate the relative concentration of
the various intracellular neutral amino acids, in particular by
transporting some amino acids out of the cell that would not be
substrates of the putative unidirectional efflux pathway (20). Because
LAT2-4F2hc efficiently exchanges intracellular
L-Cys against other extracellular neutral amino acids, this
transporter was proposed to play a major role in the basolateral efflux
of this amino acid, a part of which is produced intracellularly by the
reduction of L-cystine (16, 17, 20).
To investigate the function of the basolateral exchangers
y+LAT1-4F2hc and LAT2-4F2hc, alone,
together and in conjunction with the apical exchanger
b0,+AT-rBAT in the context of transepithelial transport, we
expressed them in MDCK cells, a recipient cell line that forms a tight
epithelium and does not express a high amount of endogenous epithelial
amino acid transporters itself.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
MDCK cells (strain II) were cultured at
37 °C and 5% CO2 in Dulbecco's modified Eagle's
medium (cat. 41965, Invitrogen, Basel, Switzerland) with 5 × 104 units/liter penicillin, 50 mg/liter streptomycin, 2 mM L-glutamine, 1% non-essential amino acids
(cat. 11140-035), Invitrogen) and 10% fetal calf serum.
Phoenix amphotropic retrovirus producer cells, kindly provided by Dr.
G. Nolan (Baxter Laboratory for Genetic Pharmacology, Dept. of
Microbiology and Immunology, Dept. of Molecular Pharmacology, Stanford University), were cultured at 37 °C and 5%
CO2 in Dulbecco's modified Eagle's medium (cat. 41966, Invitrogen, Basel, Switzerland) with 1 × 103
units/liter penicillin, 100 mg/liter streptomycin, 2 mM
L-glutamine, 1% non-essential amino acids (cat. 11140-035, Invitrogen) and 10% fetal calf serum.
cDNA Constructs, Transfection, and Retroviral
Transduction--
The MDCK cells transfected with hrBAT and
mb0,+AT were previously described (12). The
h4F2hc, mLAT2 and my+LAT1 cDNA (13, 20)
coding sequences were subcloned in the vector LZRSpBMN-Z (kindly
provided by Dr. G. Nolan) in place of the lacZ sequence. Production of
supernatants containing the pseudoviruses and subsequent transduction
of MDCK target cells was performed according to protocols provided by
Dr. G. Nolan
(www.stanford.edu/group/nolan/protocols/pro_helper_free.html), which
were adapted from Ref. 21. Briefly, Phoenix amphotropic retrovirus
producer cells were transfected with the abovementioned constructs
using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals,
Basel, Switzerland) according to the manufacturer's protocol. 16 h after transfection, the medium was exchanged, and after another
48 h at 32 °C, the supernatant was harvested and filtered. MDCK
target cells were plated at 5-10% confluency, and viral supernatants
containing 4 µg/ml polybrene (Sigma) were added 24 h later. The
freshly transduced cells were centrifuged for 30 min at 1000 rpm and
32 °C immediately thereafter. After 24 h at 32 °C the medium
was exchanged, and cells were further cultivated in normal medium at
37 °C. This procedure was repeated 8-10 times to achieve a maximal
expression of the transport proteins to be investigated.
Antibodies--
Polyclonal rabbit antibodies were raised against
synthetic peptides corresponding to the NH2 terminus of
human rBAT, MAEDKSKRDSIEMSMKGC, the COOH terminus of mouse
b0,+AT, CHLQMLEVVPEKDPE, the NH2 terminus of
mouse y+LAT1, QHEADDGSALGDGASPC, the COOH terminus of mouse
y+LAT1, CDLEDGELSKQDPKSK, and the COOH terminus of mouse
LAT2, CPIFKPTPVKDPDSEEQP, coupled to keyhole limpet hemocyanin
(Eurogentech, Seraing, Belgium). The monoclonal anti-human
4F2hc antibody used was previously described in Ref. 22.
Double Immunofluorescence Staining of Cotransfected MDCK
Cells--
Cells were seeded on filters (24-mm Corning Costar
Transwell filters, cat. Nr. 3412) at 100% confluence and cultivated
for 7 days preceding experiments. rBAT expression was induced 3 days prior to experiment with 1 µM dexamethasone. Filters were
washed three times using phosphate-buffered saline. Cells were fixed using 3% paraformaldehyde and 0.2% Triton X-100 for 15 min at room
temperature. Filters were washed three times and cut into squares.
4F2hc-rBAT, b0,+AT-4F2hc,
y+LAT1-4F2hc, and LAT2-4F2hc double
immunofluorescence was performed with a mix of the respective
antibodies 4F2hc (1:1000), rBAT (SZ564; 1:50),
b0,+AT (SZ557; 1:500), y+LAT1 (SZ398; 1:200),
LAT2 (SZ560; 1:200) in phosphate-buffered saline containing 0.5%
bovine serum albumin overnight at 4 °C. After washing, filter pieces
were incubated for 6 h at room temperature with fluorescein
isothiocyanate-labeled anti-rabbit-IgG antibody (Sigma) and CY3-labeled
anti-mouse IgG antibody (Sigma). After another round of washing, the
filters were mounted in DAKO-glycergel (DAKO, Glostrup, Denmark)
containing 2.5% 1,4-diazabicyclo (2, 2, 2) octane (DABCO) as fading
retardant. Confocal images were taken using a Leica laser scan
microscope (TCSSP, Wetzlar, Germany) equipped with a ×63 oil immersion
objective. The appropriate controls were performed without the first
and/or second primary antibodies.
Immunohistochemistry on Mouse Kidney Sections--
Kidneys of
anesthetized male mice (NMRI; RCC, Füllinsdorf, Switzerland) were
fixed for 5 min by intravascular perfusion through the abdominal aorta
as previously described (23). Coronal slices (1-2-mm thick) of the
kidney were frozen in liquid propane and stored at
80 °C until
use. Serial cryosection (4-5 µm) were cut and placed on
Chromium(III) potassium sulfate-coated glass slides. Sections were
preincubated for 10 min with 10% normal goat serum in
phosphate-buffered saline, 2% bovine serum albumin. Afterward, sections were sequentially incubated with primary antibodies against LAT2 (SZ559) 1:1000, y+LAT1 (SZ553) 1:200-1:500, and
bo,+AT (SZ400) 1:500 for 16 h at 4 °C. Binding
sites of primary antibodies were revealed with Cy3-conjugated donkey
anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).
Sections were studied by epifluorescence with a Polyvar microscope
(Reichert Jung, Vienna, Austria). For controls, consecutive
cryosections were incubated in preimmune serum. Images were acquired
with a charge-coupled device camera (Visicam 1280, Visitron System,
Puching, Germany) and processed by Image-Pro Plus version 3.0 (Media Cybernetics, Silver Spring, MD) and Corel Photoshop software.
Filter Uptake Experiments--
MDCK cells were passaged to 24-mm
Corning Costar Transwell filters at 100% confluence and cultivated for
7 days. rBAT expression was induced 24 h prior to experiment with
1 µM dexamethasone. Integrity of the monolayer was
checked by resistance measurement using the Millicell device
(Millipore, Bedford, MA). Filters were washed three times with uptake
buffer (150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2, 10 mM glucose) at
37 °C and incubated in uptake buffer for 30 min. The buffer was
replaced unilaterally with buffer supplemented with amino acid at the
indicated concentrations and the corresponding 3H-labeled
L-amino acid as tracer (except for
L-[14C]cystine); the contralateral
compartment received the same solution without the labeled
L-amino acid tracer. Uptake experiments were performed for
the times indicated. DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) (100 µM) (Sigma) was added to the solution for experiments with L-cystine. The uptake was stopped by replacing the
amino acid uptake solution with ice-cold uptake buffer and washed four times. The filters were excised and placed into scintillation vials
containing scintillation fluid (Packard, Meriden, CT). After shaking
overnight at room temperature, radioactivity was determined by
scintillation counting.
RT-PCR--
RT-PCR was performed to identify the dog LAT2
transporter. First-strand cDNA was synthesized from 100 ng of total
RNA from wild-type MDCK cells, LAT2-transfected cells serving as the
internal positive control, with or without MMRV reverse transcriptase
(Promega, Madison, WI) and 50 pmol of random hexamer primers
(Invitrogen). 1:10 of the first-strand cDNA was used as a template
for PCR amplification using 50 pmol of degenerate LAT2 primers (forward
primer 5'-GGTCAGYGCCTGTGGTATCA-3', reverse primer
5'-GCAGCACRTAGTTGGAGAAG-3' (Microsynth, Balgach, Switzerland)) and 2 units of recombinant Taq polymerase (Promega, Madison, WI).
The cycling parameters were the following: 3 min 94 °C, 35 cycles of
1 min 94 °C, 30 s 57 °C, and 1 min 72 °C, followed by 10 min at 72 °C. PCR products were separated on agarose gel, the band
cut out and extracted with QIAquick Gel Extraction kit (Qiagen, Hilden,
Germany). DNA sequencing was performed at Microsynth (Balgach,
Switzerland) using the above PCR primers.
Statistics--
Data are expressed as means ± S.E. The
difference between control and test values was evaluated using analysis
of variance (one-way) with Bonferroni's multiple comparison test.
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RESULTS |
Axial Gradient of Apical and Basolateral Heteromeric Amino Acid
Transporters along the Mouse Kidney Proximal Tubule--
The fact that
b0,+AT, LAT2 and y+LAT1 are colocalized in the
proximal tubule of the kidney in vivo is shown in Fig.
1. As yet, the in vivo
localization of y+LAT1 had not been published.
Interestingly, the present images of serial kidney sections indicate
that the localizations of b0,+AT, LAT2, and
y+LAT1 along the proximal tubule are superimposable, all
showing the same axial gradient along the proximal tubule segments
(S1>S2
S3). Thus, in view of the fact that all b0,+AT
expressed in the kidney was shown to be associated with rBAT (24), the
three heteromeric transporter b0,+AT-rBAT,
LAT2-4F2hc, and y+LAT1-4F2hc
colocalize along the kidney proximal tubule with a very similar axial
gradient. Only for y+LAT1, the staining along the S1 and S2
segments appears to terminate earlier. Interestingly, some LAT2
staining is visible along the distal tubules as well.

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Fig. 1.
Localization of b0,+AT, LAT2, and
y+LAT1 in the mouse kidney. Serial cryosection (4-5
µm) of mouse kidney were stained with antibody against
b0,+AT (left), y+LAT1
(middle), and LAT2 (right). Staining of the
kidney cortex (top) revealed that all three transporters are
colocalized in proximal tubule cells, showing the same gradient of
expression, from S1>S2 S3 segment. A larger magnification view
(bottom) shows that expression of all three proteins
commences directly after the glomerulus. The signal for
b0,+AT is localized in the brush border membrane, whereas
both y+LAT1 and LAT2 are found in the basolateral membrane
of the same cells. Scale bar: top, 80 µm;
bottom: 20 µm. G, glomerulus; D,
distal tubule, S1, S1 segment of the proximal tubule.
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Basolateral Localization of the Heteromeric Transporters
y+LAT1-4F2hc and LAT2-4F2hc in Transduced MDCK
Cells--
To perform a functional analysis of coexpressed heteromeric
amino acid transporters of the proximal tubule and intestine, the MDCK
cell line that is of distal nephron origin was chosen as recipient,
because it does not express a high baseline transepithelial amino acid
transport activity. Nevertheless, for the interpretation of the data
obtained on transfected/transduced MDCK cells, one has to keep in mind
that they do express some endogenous transporters. For instance,
experiments performed in the eighties suggested the presence of
endogenous system A and L (mostly basolateral), ASC (basolateral and
apical), and of an apical Na+/amino acid symporter with a
broad substrate selectivity (25).
The MDCK cell line already expressing the apical heteromeric
transporter b0,+AT-rBAT as well as untransfected MDCK cells
were sequentially transduced with amphotropic pseudoretrovirus encoding
the subunits of the heteromeric transporters
y+LAT1-4F2hc and/or LAT2-4F2hc. The
steady-state localization of these gene products was then analyzed by
immunofluorescence confocal microscopy. Fig.
2, A and B shows
that 4F2hc, when expressed with LAT2 in
b0,+AT-rBAT-expressing cells, localizes mainly to the
lateral membrane. Only little intracellular and no apical
4F2hc staining can be seen. At the cell surface, there is
also no overlap with rBAT (panel A, apical staining) or
b0,+AT (panel B, intracellular and apical
staining).

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Fig. 2.
Colocalization of rBAT, b0,+AT,
LAT2, or y+LAT1 with 4F2hc in transfected
MDCK cells. Images taken parallel to the filter (X-Y plane) at the
level of the nucleus are shown in the square panels. The
rectangular panels represent corresponding Z-Y
reconstitutions. Scale bar: 10 µm. A, in MDCK
cells transfected with b0,+AT-rBAT and
LAT2-4F2hc, rBAT (green) shows a strict apical
localization, while 4F2hc (red) is found in the
basolateral membrane. B, visualization of b0,+AT
(green) and 4F2hc (red) in the same
cells show the same basolateral localization for 4F2hc,
while b0,+AT is found intracellularly in large excess and
in the apical membrane. C, in cells coexpressing LAT2
(green) and 4F2hc (red), both proteins
colocalize to the basolateral membrane, as seen by the yellow
staining (Z-X reconstitution). D, cells coexpressing
y+LAT1 (green) and 4F2hc
(red) show the same basolateral colocalization of both
proteins (colocalization yellow). Note that in panels
C and D some cells exhibit, besides intracellular
staining, a basolateral staining for LAT2/y+LAT1 in
the absence of 4F2hc staining, suggesting the presence of
low amounts of endogenous 4F2hc.
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LAT2 and 4F2hc co-localize in the basolateral membrane (Fig.
2C), as expected from their localization in mouse kidney and small intestine (20). In this figure, made at a stage at which only
~50% of the cells expressed both 4F2hc and LAT2, some
cells exhibited LAT2 staining only, that, interestingly, was localized both intracellularly and at the lateral membrane. Because LAT2 was
previously shown to necessitate association with 4F2hc for functional surface expression in Xenopus oocytes, the
partial membrane localization of LAT2 in the absence of visible
4F2hc indirectly suggests the presence of some endogenous
canine 4F2hc that is not recognized by the species-specific
monoclonal antibody. The fact that coexpression of exogenous
4F2hc leads to an essentially basolateral immunolocalization
of LAT2 confirms the hypothesis that 4F2hc is the limiting
factor for LAT2 surface expression in MDCK cells.
The subcellular localization of y+LAT1 has been visualized
as yet only in Xenopus oocytes and in non-polarized
transfected HEK293 cells (26, 27). Here we demonstrate that exogenous y+LAT1 behaves as LAT2, namely that it colocalizes with
exogenous 4F2hc in the basolateral membrane of MDCK cells
(Fig. 2D) and that in the absence of exogenous
4F2hc, only a small fraction of it appears at the
basolateral surface (putatively associated with endogenous
4F2hc).
Cooperation of Apical and Basolateral Heteromeric Amino Acid
Transporters for L-Arg Transport--
To test the role and
the cooperation of b0,+AT-rBAT and/or
y+LAT1-4F2hc on transepithelial cationic amino
acid transport, we added L-Arg and L-Leu
together to both sides of the epithelia, to allow amino acid exchange
to proceed in the absence of a transepithelial concentration gradient.
L-Arg was chosen because it is, on the one hand, a good
influx substrate for the apical transporter b0,+AT-rBAT
that exchanges preferentially extracellular cationic amino acids or
L-cystine against intracellular neutral amino acids and, on
the other hand, a good efflux substrate for the basolateral transporter
y+LAT1 known to preferentially exchange intracellular
cationic amino acids against extracellular neutral amino acids plus
Na+ (13, 14). L-Leu was taken as second
substrate because it is a good influx substrate for y+LAT1
and a suitable efflux substrate for b0,+AT-rBAT (5, 13,
14).
Radioactive tracer of either amino acid was added on separate filter
cultures to each side of the epithelia and the amount of labeled amino
acid in the cell and at the contralateral side was measured after a 2-h
incubation. Because of interexperimental variation in absolute
transport levels, amounts of accumulated amino acids were arbitrarily
normalized for each experiment to the amount of L-Arg taken
up from the apical side by b0,+AT-rBAT-expressing cells
(mean: ~0.8 nmol × cm
2 × 2h
1).
MDCK cells expressing no b0,+AT-rBAT exhibited almost no
apical uptake of L-Arg and displayed only a small
apical-to-basolateral (A to B) flux (Fig.
3A). In contrast, when
b0,+AT-rBAT was expressed at the apical membrane, the
intracellular amount of L-[3H]Arg was
increased by a factor of ~30. With epithelia expressing no exogenous
basolateral transporter, an amount corresponding to 2.7× the
intracellular L-Arg was accumulated in the basolateral chamber within 2 h, suggesting that there is an endogenous
basolateral efflux pathway for L-Arg in MDCK cells.
However, the additional expression of basolateral
y+LAT1-4F2hc led to a further increase in
basolateral L-[3H]Arg accumulation and to a
slight decrease in its intracellular level such that the ratio of
basolateral/intracellular L-[3H]Arg was
increased by a factor of two, indicating that
y+LAT1-4F2hc mediates the basolateral efflux of
L-Arg.

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Fig. 3.
Cooperation of apical and
basolateral heteromeric amino acid transporters for L-Arg
and L-Leu transport. Cells expressing combinations of
heteromeric amino acid transporters, indicated by the individual
abbreviations (key in A, bottom left panel), were
cultivated on permeable supports. The filter cultures were incubated
for 2 h with 50 µM L-Arg and
L-Leu +Na+ on both sides of the monolayer, with
the tracer L-[3H]Arg (A) or
L-[3H]Leu (B) added either to the
apical (1st row) or the basolateral (2nd row)
compartment. After the 2-h period, aliquots of the contralateral
compartment (apical (A) or basolateral (B)) were
taken to determine the unidirectional transcellular transport, and the
cells lysed to determine the cellular (C) accumulation. The
net transport rate across the monolayer (3rd row) is the
difference between the unidirectional transport rates. The amounts of
transported amino acids in individual experiments were arbitrarily
normalized to the amount of L-Arg accumulated
intracellularly from the apical side by
b0,+AT-rBAT-expressing cells (mean: ~0.8 nmol × cm 2 × 2h 1), n = 9;
error bars, S.E.; **, p < 0.01; ***,
p < 0.001.
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Wild-type MDCK cells displayed an efficient basolateral uptake of
L-Arg, probably via a y+-type transporter.
However, the apical L-Arg accumulation was small,
indicating that there is no efficient endogenous apical efflux pathway
for L-Arg. The expression of the basolateral transporter LAT2-4F2hc and/or y+LAT1 in wild-type MDCK cells
did not change the basolateral uptake of L-Arg.
All cells expressing apical b0,+AT-rBAT displayed a higher
basolateral-to-apical flux of L-Arg that is compatible with
the hypothesis that b0,+AT-rBAT transports some
L-[3H]Arg apically out of the cells, possibly
by homoexchange against unlabeled apical L-Arg. The fact
that there was also an increase in cellular
L-[3H]Arg imported from the basolateral side
can be attributed to a transtimulatory effect of the apically imported
unlabeled L-Arg at the level of the endogenous basolateral
y+ transporter (homoexchange). This hypothesis is supported
by the fact that in the cell lines that express basolateral
y+LAT1 (that prefers heteroexchange of intracellular
L-Arg for extracellular L-Leu + Na+), the increase in labeled intracellular
L-Arg was much less pronounced.
The resulting net L-Arg transport is in the
apical-to-basolateral direction. The effect of b0,+AT-rBAT
expression is such that more L-Arg is being transported into the cell and thus also extruded through the basolateral membrane. This extrusion is increased by the presence of
y+LAT1-4F2hc in the basolateral membrane.
The apical uptake of L-Leu (intracellular + basolateral
accumulation) is significantly increased by the expression of
b0,+AT-rBAT, confirming that this exchanger does transport
L-Leu also inwards, though less efficiently than it does
L-Arg (Fig. 3B). This experiment also shows that
MDCK cells display an efficient endogenous basolateral efflux pathway
for L-Leu, since the ratio of labeled basolateral to
intracellular L-Leu was high. It is noteworthy that neither
the addition of LAT2-4F2hc nor of
y+LAT1-4F2hc increased the basolateral efflux of
L-Leu, an observation that is not surprising in view of the
fact that L-Leu is not a good efflux substrate for either
of these exchangers. The apical extrusion of L-Leu was
increased by the expression of b0,+AT-rBAT, demonstrating
that L-Leu is an efflux substrate for this exchanger. The
fact that this apical extrusion was increased by the expression of
either basolateral exchanger (significant only in the case of
coexpression of both transporters) strongly supports the notion that
both LAT2-4F2hc and y+LAT1-4F2hc
import L-Leu from the basolateral side.
The net L-Leu transport across the wild type MDCK monolayer
is in an apical-to-basolateral direction ((re)absorption, compatible with the presence of some apical Na+-neutral amino acid
transport activity (25)) and unchanged by the expression of either the
apical or the basolateral heteromeric transporters. However, if a
combination of apical b0,+AT-rBAT and either basolateral
transporter is expressed, the direction of the net L-Leu
transport is reversed into the basolateral-to-apical direction. In the
case of y+LAT1-4F2hc expression, the change in
L-Leu net flux is of the same magnitude but opposite
direction than that of L-Arg, indicating that this
transporter cooperates with apical b0,+AT-rBAT for a
transepithelial exchange of L-Arg against
L-Leu. The effect of LAT2-4F2hc on apical
L-Leu efflux does not correspond to an increase in
apical-to-basolateral L-Arg flux and can thus be explained
by a basolateral exchange of extracellular labeled L-Leu
against endogenous neutral amino acids.
Cooperation of Apical and Basolateral Heteromeric Amino Acid
Transporters for L-Cystine Transport--
The role of
b0,+AT-rBAT and of its cooperation with basolateral
LAT2-4F2hc and/or y+LAT1-4F2hc for
transepithelial transport of L-cystine was tested by adding
to both sides the amino acid pair L-cystine + L-Leu (Fig. 4A).
The transport of L-cystine was tested because it is its
tubular accumulation that leads to most clinical problems found in
cystinuria. Concerning the basolateral efflux of L-cystine, it is believed that its reduction products (two L-Cys) are
extruded, possibly by the exchanger LAT2 that efficiently exports
L-Cys when expressed in Xenopus oocytes
(15).

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Fig. 4.
Cooperation of apical and basolateral
heteromeric amino acid transporters for L-cystine and
L-Leu transport. Cells expressing combinations of
heteromeric amino acid transporters, indicated by the individual
abbreviations (key in A, bottom left panel), were
cultivated on permeable supports. The filter cultures were incubated
for 2 h with 100 µM L-cystine and
L-Leu +Na+ on both sides of the monolayer, with
the tracer L-[14C]cystine (A) or
L-[3H]Leu (B) added either to the
apical (1st row) or the basolateral (2nd row)
compartment. After the 2-h period, aliquots of the contralateral
compartment (apical (A) or basolateral (B)) were
taken to determine the unidirectional transcellular transport, and the
cells lysed to determine the cellular (C) accumulation. The
net transport rate across the monolayer (3rd row) is the
difference between the unidirectional transport rates. The amounts of
transported amino acids in individual experiments were arbitrarily
normalized to the amount of L-cystine accumulated
intracellularly from the apical side by
b0,+AT-rBAT-expressing cells (mean: 0.52 nmol × cm 2 × 2h 1 of L-cystine,
corresponding to 1.04 nmol × cm 2 × 2h 1 L-Cys), n = 9; error
bars, S.E.; **, p < 0.01; ***, p < 0.001.
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Similar to above, we arbitrarily normalized the flux data of each
experiment to the amount of L-cystine/L-Cys
(counted as L-cystine) that was accumulated intracellularly
from the apical side in cells expressing b0,+AT-rBAT only.
This amount was in average 0.52 nmol × cm
2 of
L-cystine (corresponding to 1.04 nmol × cm
2 × 2h
1 L-Cys), which is of
the same range as that measured for L-Arg (0.8 nmol × cm
2 × 2h
1).
In wild-type MDCK cells, L-cystine was apically taken up
more efficiently than L-Arg but less than
L-Leu. However, its basolateral efflux (as
L-cystine and/or two L-Cys) was less efficient
than that of the two other substrates (lower basolateral/intracellular ratio) and was not increased by the expression of the basolateral transporters. The expression of apical b0,+AT-rBAT led to a
4-fold higher intracellular accumulation of L-cystine, as
expected from our previous study (12). Its basolateral extrusion (probably as L-Cys) was increased as well, though by a
lower factor. The coexpression of either basolateral transporter
increased the basolateral extrusion of
L-cystine/L-Cys. As mentioned above, this was
expected for LAT2-4F2hc that can exchange intracellular L-Cys for extracellular L-Leu (15). However,
y+LAT1-4F2hc was not expected to mediate
L-cystine or L-Cys efflux. Thus, we tested the
possibility that y+LAT1 was an efflux pathway for
L-Cys in Xenopus oocytes but did not observe any
transport (not shown). We then tested the hypothesis that MDCK cells
express LAT2 endogenously. This would explain the increase in
basolateral L-Cys efflux upon expression of exogenous 4F2hc, assuming that endogenous 4F2hc would limit
(endogenous) LAT2 surface translocation. With a set of degenerate
primers we identified a partial sequence from MDCK cells bearing 89%
identity with the mouse, 90.5% identity with the rat, and 91.5%
identity with the human sequences. This suggests that the expression of 4F2hc alone (or with y+LAT1) induces the
expression/activation of the endogenous LAT2 transporter, which is
responsible for the observed L-Cys efflux. This hypothesis
was corroborated by the fact that expression of 4F2hc
alone increased cystine reabsorption in
b0,+AT-rBAT-expressing cells (not shown).
Because the basolateral-to-apical transport of L-cystine
was not affected, in contrast to its apical-to-basolateral transport, by any of the exogenous transporters, net transport of
L-cystine (that was close to zero in wild-type MDCK cells)
was strongly increased in the apical-to-basolateral direction by the
expression of apical b0,+AT-rBAT and further increased by
the coexpression of either basolateral exchanger.
In the same series of experiments, the transport of L-Leu
was qualitatively affected in a similar way by the expression of heteromeric amino acid transporters as in the experiments in which it
was given together with L-Arg (see above). However, due to the unfavorable ratio of exogenous to endogenous transport and the
experimental variability, changes in net L-Leu transport
did not reach the level of significance.
 |
DISCUSSION |
Early Proximal Tubule as Major Expression Site of the Three Amino
Acid Exchangers That Form a Cationic Amino Acid and Cystine Transport
Module--
The present immunolocalization performed on mouse kidney
sections demonstrates that the three gpaATs b0,+AT, LAT2,
and y+LAT1 are coexpressed in cells of the proximal tubule,
following exactly the same axial gradient (Fig. 1). They are strongly
expressed in the S1>S2 segments and display a much weaker expression
in S3. Former studies have already described the kidney brush border localizations of b0,+AT and its subcellular colocalization
with rBAT as well as the fact that rBAT displays an opposite axial
distribution gradient along the proximal tubule (S3
S2>S1) (13).
The basolateral localization of LAT2 and its colocalization with
4F2hc were also previously illustrated (20). We now show
that y+LAT colocalizes with LAT2 in the basolateral
membrane of the same kidney proximal tubule cells that also express
apical b0,+AT and thus follows the same axial gradient.
This precise coexpression of three gpaATs suggests that they function
together as a building block of the transcellular amino acid
reabsorption machinery for cationic amino acids and cystine. The
parallelity of y+LAT1 and LAT2 expression with that of
b0,+AT, and not with rBAT, implies that the reabsorption of
these amino acids follows the same proximal to distal gradient, and suggests that the additional high level expression of rBAT in the S3
portion of the proximal tubule is not linked to an additional dibasic
amino acid reabsorption. The fact that all kidney b0,+AT
appears to be associated with rBAT has been recently shown and supports
the hypothesis of an additional rBAT function in the S3 segment
(24).
Serial b0,+AT-rBAT and y+LAT1-4F2hc Are a
Functional Unit for Cationic Amino Acid (Re)absorption--
The
expression of the b0,+AT-rBAT transporter leads to apical
L-Arg influx in MDCK cells, as previously demonstrated
(12). In the absence of added basolateral transporter, this already leads to a net transepithelial transport of L-Arg (Fig.
3A). It appears likely that in this case the basolateral
efflux of L-Arg is mediated by an endogenous
y+-type and not by a y+L type transporter,
since L-Arg is not exchanged against extracellular L-Leu. L-Arg uptake by system y+
(CAT transporters) is known to be trans-stimulated by intracellular cationic amino acids and thus obeys a non-obligatory exchange mode
(uniport) (see Ref. 28 for review). Thus, at the low extracellular y+ substrate concentration used in this study (50 µM L-Arg), a favorable concentration gradient
appears to overcome the electrical gradient and thus permits the net
efflux of L-Arg via a basolateral CAT transporter.
Here, we show that the additional expression of
y+LAT1-4F2hc in the basolateral membrane
increases the transepithelial transport of L-Arg and, by
the same amount, the opposite transport of L-Leu. This is
the clear demonstration that the in-series arrangement of the two
obligatory exchangers b0,+AT and y+LAT1
suffices to mediate transepithelial L-Arg (re)absorption, when an exchange substrate and Na+ are present. At the
apical membrane, the influx of L-Arg is thus driven by the
membrane potential and is coupled to the efflux of a neutral amino
acid, in this case essentially L-Leu (the only neutral
amino acid present in the extracellular milieu). However, the
relatively low ratio of apical/intracellular labeled L-Leu suggests that L-Leu might not be a high affinity substrate
for efflux via b0,+AT-rBAT. L-Arg taken up
apically by b0,+AT is sequentially transported across the
basolateral membrane via y+LAT1 in exchange for
L-Leu and Na+ (additionally to an efflux via
endogenous y+; see above). This electroneutral transport is
driven by the outward-directed concentration gradient of
L-Arg and inward one of Na+. The fact that in
the presence of y+LAT1 some transepithelial
L-Arg transport that is not compensated by an opposite
L-Leu transport persists, although the driving forces at
the basolateral side should favor the latter transport over the
endogenous y+-type one, can be explained by the fact that
not all b0,+AT-rBAT-expressing cells express a
corresponding level of basolateral y+LAT1-4F2hc
(see inhomogeneity in Fig. 2).
The serial b0,+AT-rBAT and
y+LAT1-4F2hc exchangers thus represent a
functional unit for the reabsorption of cationic amino acids that, in
exchange, secretes neutral amino acids (in this case L-Leu). Thus, to assure a net reabsorption of neutral amino
acids as well, this functional unit needs to be installed in parallel with a unidirectional transcellular (re)absorptive pathway for neutral
amino acids, in particular for the exchange substrates of
b0,+AT and y+LAT1-4F2hc. The major
apical transport system that corresponds to those criteria is
B0 that, however, has not been yet molecularly defined. At
the basolateral membrane, the characteristics of an efflux pathway for
neutral amino acids have not yet been described.
Role of LAT2-4F2hc for
L-cystine/L-Cys
Reabsorption--
For L-cystine (maintained
extracellularly in oxidized form with DTNB and presumably reduced to
two L-Cys intracellularly), as for L-Arg, the
apical expression of b0,+AT-rBAT not only increases the
apical uptake, but also the net transepithelial flux, even in the
absence of an additional basolateral transporter. It appears that an
endogenous basolateral pathway mediates this basolateral
L-Cys efflux in a unidirectional manner, because there is
no concomitant increase in basolateral L-Leu or
L-cystine uptake (the only two amino acids present
extracellularly in these experiments). The nature of the pathway that
mediates this unidirectional efflux of L-Cys is not known.
The existence of such a pathway could play a role in maintaining the
cellular osmolyte level, because the intracellular reduction of
L-cystine into two L-Cys increases the number
of intracellular solute molecules.
The additional expression of 4F2hc alone or in combination
with LAT2 or y+LAT1 yielded a small but significant
increase in the basolateral extrusion of
L-cystine/L-Cys in
b0,+AT-rBAT-expressing cells. The fact that
4F2hc alone already had this effect (data not shown)
suggests that 4F2hc activates an endogenous
L-Cys efflux pathway. We tested by RT-PCR the hypothesis that LAT2 is the activated endogenous transporter because LAT2 is known
from Xenopus laevis oocyte expression experiments
to mediate the efflux of L-Cys when coexpressed with
4F2hc (15). The presence of a corresponding transcript in
MDCK cells suggests indeed that exogenous 4F2hc increases
the surface expression of endogenous LAT2 that otherwise is limited by
the low level of endogenous 4F2hc.
Thus, although the present data lack the clarity of those obtained for
L-Arg transport by y+LAT1, due to the
expression of an endogenous LAT2 and of the relatively high transport
rates of L-Leu by endogenous transporter(s) that prevent a
precise quantification of basolateral L-Cys - L-Leu exchange, they strongly suggest that LAT2
participates in basolateral L-Cys efflux in the context of
transepithelial L-cystine/L-Cys transport.
These data also support the notion that MDCK cells express a
basolateral unidirectional efflux pathway for neutral amino acids that
can function in parallel with LAT2-4F2hc and that is
required for net directional amino acid transport across the
basolateral membrane. Whether classical L-Cys transporters like asc1 (4F2hc-associated transporter) or ASCT2 are
involved in this efflux remains to be established.
Endogenous Transporters in MDCK Cells--
As shown by RT-PCR and
suggested by the functional experiments discussed above, MDCK cells
possess endogenous LAT2, a part of which might be functionally
expressed in the absence of exogenous 4F2hc. This would
imply the presence of some endogenous 4F2hc, a possibility
that is supported by the immunolocalization study that shows a partial
basolateral membrane localization of y+LAT1 and/or LAT2 in
the absence of exogenous 4F2hc.
The functional experiments furthermore demonstrate that endogenous
transport systems for influx and efflux of L-Leu exist in
both the apical and basolateral membranes of MDCK cells. The (re)absorptive transport of L-Leu observed across
untransfected MDCK epithelia is compatible with the previous
observation of a Na+-dependent neutral amino
acid transport that resembles B0 (25). The fact that most
L-Leu taken up apically via b0,+AT-rBAT is
extruded basolaterally also in the absence of exogenous basolateral
transporter indicates that MDCK cells express an endogenous basolateral
efflux system for L-Leu that has apparently a high capacity
and possibly also a relatively high affinity. The molecular nature of
the transporter that mediates this extrusion of L-Leu and
whether it is the same one that transports L-Cys remains to be established.
In conclusion, the present study demonstrates the cooperation of apical
and basolateral heteromeric exchangers for the transepithelial transport of cationic amino acids and L-cystine and the
fact that these transporters also function as exchangers in an
epithelial context. The opposite direction of neutral amino acid
transport (exchange) supports the notion that additional unidirectional transporters of neutral amino acids need to be expressed in both the
apical and the basolateral membrane of directionally transporting epithelia to recycle the exchange substrates and thus drive the transport of neutral amino acids in the (re)absorptive direction. Such
an apical unidirectional transport system has been functionally characterized in the kidney proximal tubule (B0) but not
yet molecularly identified. The basolateral unidirectional efflux
pathway, that appears to be expressed at a certain level also in MDCK
cells, remains to be further characterized.