(Received for publication, November 28, 1995; and in revised form, February 12, 1996)
From the
Several studies have shown that the cRNA of human, rabbit, or
rat rBAT induces in Xenopus oocytes sodium-independent, high
affinity uptake of L-cystine via a system
b-like amino acid exchanger. We have shown that
mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini,
P., Chillarón, J., Chillón,
M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola,
B., Di Silverio, F., Barceló, P., Estivill, X.,
Zorzano, A., Nunes, V., and Palacín,
M.(1994) Nat. Genet. 6, 420-425; Calonge, M. J.,
Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E.,
Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P.,
Nunes, V., and Palacín, M.(1995) Proc.
Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from
oocytes, no other expression system has been used for transfection of
functional rBAT activity. Furthermore, the b
-like
transport activity has not been clearly described in the kidney or
intestine. Here, we report that a ``proximal tubular-like''
cell line derived from opossum kidney (OK cells) expresses an rBAT
transcript. Poly(A)
RNA from OK cells induced system
b
-like transport activity in oocytes. This was
hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase
chain reaction-amplified cDNA fragment (
700 base pairs) from OK
cell RNA corresponds to an rBAT protein fragment 65-69% identical
to those from human, rabbit and rat kidneys. We have also examined
transport of L-cystine in OK cells and found characteristics
very similar to the amino acid exchanger activity induced by rBAT cRNA
in oocytes. Uptake of L-cystine was of high affinity,
sodium-independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the
same specificity as rBAT-induced transport activity in oocytes.
Furthermore, it was localized to the apical pole of confluent OK cells.
To demonstrate that the rBAT protein is functionally related to this
transport activity, we have transfected OK cells with human rBAT
antisense and sense sequences. Transfection with rBAT antisense, but
not with rBAT sense, resulted in the specific reduction of rBAT mRNA
expression and b
-like transport activity. These
results demonstrate that rBAT is functionally related to the L-cystine uptake via system b
-like in the
apical pole of the renal OK cell line.
Human rBAT cDNA elicits high affinity sodium-independent
transport of cystine, dibasic amino acids, and some zwitterionic amino
acids via a b-like transport system in Xenopus oocytes(1, 2) . Heteroexchange diffusion of these
amino acids has been reported for this transport activity expressed by
rabbit(3, 4) , rat(5) , and human (
)rBAT cRNA in oocytes. rBAT protein is expressed in the
brush border plasma membrane of both the proximal straight tubules of
the nephron and the small intestine(6, 7) . Recent
studies have demonstrated that mutations in the human rBAT gene cause
cystinuria(8, 9, 10, 11, 12) .
Cystinuria is a common inherited aminoaciduria due to the defective
transport of cystine and dibasic amino acids through the epithelial
cells of the renal tubule and intestinal
tract(13, 14) . The clinical manifestation of
cystinuria is the development of kidney cystine calculi resulting from
the poor solubility of this amino acid(14) . Three types of
classic cystinuria have been described on the basis of the amino acid
hyperexcretion of heterozygotes and the degree of the intestinal
transport defect(14, 15, 16) . Very recently,
strong evidence has been offered suggesting that rBAT is only
responsible for type I(10, 12) .
Due to the role of
the rBAT gene in type I cystinuria, the rBAT protein is
considered to be responsible for the reabsorption of cystine and
dibasic amino acids in the proximal straight tubule. Two aspects of
this remain to be clarified. First, a complete picture of the
b-like transport activity responsible for cystine
reabsorption has not been described in the epithelial cells of kidney
or intestine. In renal brush border membrane vesicles a high affinity
system for cystine and dibasic amino acids, which shows heteroexchange
diffusion between these substrates, has been
demonstrated(17, 18, 19) . This transport was
shown to be defective in biopsies of intestinal mucosa from cystinuric
patients(20, 21, 22) . The sodium dependence
of this reabsorption system is controversial, as is its interaction
with neutral amino acids, since no hyperexcretion of neutral amino
acids occurs in
cystinuria(17, 18, 19, 22, 23, 24) .
Second, the role of rBAT in the b
-like transport
mechanism is unknown. Evidence from Tate and co-workers (25) suggests a four-membrane-spanning domain model for rBAT.
This topology is unusual for metabolite transporters, which appear to
contain 8-12 transmembrane domains(26) . This fostered
the idea that rBAT is a regulatory subunit of an oligomeric transporter
rather than the transporter itself(27, 28) . Indirect
evidence suggested that rBAT forms a heterodimeric structure of 125 kDa
with an unidentified protein of 40-50 kDa in renal brush border
membranes and oocytes(29) . (
)In addition, transient
expression of rBAT in COS cells revealed either expression of rBAT in
the cell surface without concomitant amino acid transport activity (30) or a protein product that does not reach the plasma
membrane. (
)Thus, no cell system other than oocytes has
shown expression of amino acid transport activity associated with rBAT.
It thus remains an open question whether the amino acid transport
activity associated with rBAT in the renal epithelial cells is the same
as that elicited by rBAT expression in oocytes.
To this end we
searched for the role of rBAT in the transport of cystine in an
epithelial cell line derived from proximal tubules of the kidney of an
American opossum, the cell line OK. These cells express an mRNA
transcript that hybridizes with rBAT probes(27, 28) ,
and they show high affinity sodium-independent cystine-dibasic amino
acid transport in the apical pole(31) . Here we demonstrate
that OK cells express rBAT mRNA and that the transport of cystine in
the apical pole of these cells is due to an amino acid transport system
very similar to the b-like system elicited by rBAT
in oocytes. Transfection of rBAT antisense sequences results in the
specific reduction of rBAT mRNA expression and
b
-like transport activity. This demonstrates that
rBAT is responsible for the high affinity sodium-independent L-cystine uptake shared with dibasic and neutral amino acids (i.e. system b
-like) present in the apical
pole of the renal cell line OK.
For transport studies, cells
were used 5 days after plating (90-100% confluence) on 35-mm
diameter dishes, except for antisense studies, in which 16-mm diameter
wells were used. Growth medium was removed, and dishes were washed
three times in 3 ml of MGA ()medium (i.e. 137
mMN-methyl-D-glucamine (MGA), 5.4 mM KCl, 2.8 mM CaCl
, 1.2 mM MgSO
, 10 mM HEPES; pH 7.4 at 37 °C)
prewarmed to 37 °C. MGA was replaced by 137 mM NaCl
(sodium medium) in those experiments in which the sodium-dependence was
studied. Uptake media were prepared by adding the labeled amino acid (L-[
H]arginine and L-[
H]leucine from DuPont NEN or L-[
S]cystine from Amersham Corp; final
concentration, 0.5-1 µCi/ml) to MGA or sodium media. When
cystine was present, the uptake medium contained 5 mM diamide
as an oxidizing agent. Uptake was started by the addition of 1 ml (0.5
ml for 16-mm diameter wells) of uptake medium (at 37 °C) to the
plate and terminated by removing uptake medium from the plate and
washing it five times in cold stop solution (132 mM NaCl, 14
mM Tris/HCl, 5 mML-arginine, 5 mML-leucine; pH 7.4 at 4 °C). For the three amino acids
used, uptake periods were previously assessed for all concentrations
studied and consequently the uptake period used was 30 s. Nonspecific
binding was assessed by measuring zero-time uptake, which was achieved
by adding the uptake medium and immediately removing it and stopping
the uptake. Cell lysates were obtained by adding 1 ml of 0.5% Triton
X-100/plate (0.5 ml in 16-mm diameter wells). 150 µl of this lysate
was removed for scintillation counting in 3 ml of
EcoLite
(TM) scintillation fluid (ICN), and 25
µl was used for protein determination by the Lowry
method(34) . The zero point was subtracted from the 30-s value,
and uptake is expressed as nmol/mg protein
min. Previous studies
demonstrated that after subtracting the zero value, the best fit line
passed through the origin (data not shown).
For trans-stimulation
experiments, cells in a confluent 3.5-cm diameter plate were preloaded
for 2 min with 50 µML-[H]arginine or L-[
H]leucine (0.5 µCi/ml).
Afterward, they were washed in MGA medium and 1 ml of MGA prewarmed
medium was added to the plate. From 15 s to 10 min, the efflux was
monitored by removing aliquots (50 µl) periodically from the
medium. The radioactivity contained in each aliquot was quantified by
liquid scintillation counting. Results are expressed as (counts/min)/mg
of protein/min. Efflux was linear for up to 1 min. Thin layer
chromatography of the efflux medium in silica gel using butanol/acetic
acid/water (4:1:1, by volume) as a solvent, as described
elsewhere(35) , revealed that
95% of the efflux
radioactivity corresponds to L-arginine.
For transport polarity studies, OK cells were grown on Handmade transwell filters (Nucleopore Corp. filtration products). A 1-cm diameter polycarbonate tube was cut in 1-cm sections, and polycarbonate filter membrane (0.1-µm pore size, 12-cm diameter, Nucleopore) was glued with 1:1 cyclohexanone:chloroform to one side of each section. The filters were dried, sterilized in 70% ethanol for at least 2 h, dried overnight, and collagen-coated with (50 µl/filter) of rat tail collagen (R-type, Serva; 0.5 mg/ml in 50% ethanol). Cells were seeded at high density 1/1.3 from the final trypsinized volume (20 ml of cell suspension). 500 µl of medium was added to the basal surface. Cells were refed 1 day before the uptake, which was carried out 3 days after the seeding, when cells had reached confluence. Transport measurements were performed using the uptake medium described above for plastic cell dishes. Both sides of the plastic transwell filters were thoroughly washed in MGA medium. For apical transport, in either the presence or absence of sodium, basal side was immersed in MGA medium. Uptake was initiated by the addition of 500 µl of the uptake medium to the corresponding side of the filter. For basal transport, the same procedure was followed, i.e. the apical side was immersed in MGA medium. Uptake was stopped by aspirating the uptake solution and washing the filters five times in the stop solution mentioned above. 100 µl of 0.5% Triton X-100 was added to each filter, and, 30 min later, total radioactivity incorporated into the monolayer was measured by liquid scintillation counting of the whole filter immersed in 9 ml of scintillation fluid. Results are expressed as pmol/30 s/filter.
For hybrid depletion experiments, OK cell mRNA (0.7-1.0 mg/ml) or human rBAT cRNA (0.01 mg/ml), prepared as described elsewhere(1) , were denatured at 65 °C for 5 min in a solution containing 50 mM NaCl and 20 µM of a 20-mer sense or a 21-mer antisense oligonucleotide complementary to the human (and rat) rBAT mRNA sequence (sense: 5`-TGC CCA AGG AGG TGC TGT TC-3`, starting at base 203 of the coding region; antisense: 5`-GAA CAG CAC CTC CTT GGG CAT-3`, starting at base 222 of the coding region) and further incubated at 42 °C for 30 min prior to oocyte injection. In these conditions, human rBAT cRNA-induced amino acid transport activity was specifically hybrid depleted by >80% by the above mentioned antisense oligonucleotide (data not shown).
The purified C4D-C5R PCR-fragment was sequenced with the same primers as for amplification and the internal primers C4R and C5D, described elsewhere(8) , using an automatic DNA sequencer (Applied Biosystems model 373A) and Taq DyeDeoxy(TM) terminator cycle sequencing kit.
For transfections and clone
selection, confluent OK cell monolayers (day 0) were trypsinized and
seeded 1/7 in two 25-cm flasks. On day 1 (40-50% of
confluence) cells were washed three times and then covered with 3 ml of
serum-free medium. Cells were then transfected by adding dropwise 120
µl of DNA-Lipofectin mixture to each flask and swirling gently.
DNA-Lipofectin mixture (1:1, v/v) was prepared with human rBAT sense or
antisense constructs (45 µg/60 µl) and Lipofectin (30 µg/60
µl), following supplier's protocol (Life Technologies, Inc.).
Cells were incubated with DNA-Lipofectin-containing medium for 16 h in
a humidified atmosphere of 5% CO
, 95% air at 37 °C.
Medium was then removed and replaced by complete medium (i.e. with 10% serum). Cells were grown to confluence, trypsinized, and
seeded in the presence of 0.4 mg/ml Geneticin (G418; Life Technologies,
Inc.) to a very low density (1/200) so that single clones could be
isolated by picking the clones with sterile trypsin-embedded
cotton-sticks. G418-resistant clones were continuously grown in the
presence of G418 (0.4 mg/ml). Screening of positive clones expressing
the sense or antisense human transcript was performed by Northern blot
analysis using BamHI fragment of human rBAT cDNA cloned in
pSPORT1 (1) .
Figure 1:
rBAT antisense hybrid depletion of the L-arginine-inhibitable L-leucine uptake induced by OK
cells mRNA in oocytes. Oocytes were injected with water containing 0 or
35 ng of OK cell mRNA. Prior to injection, mRNA was incubated alone (Control) or in the presence (20 µM) of a human
rBAT antisense or sense oligonucleotide. Five days after injection,
uptake of 50 µML-leucine was determined for 30
min incubations and either in the absence or in the presence of 5
mML-arginine. Each data point is the mean ±
S.E. of the values (pmol/30 min/oocyte) of OK cell mRNA-induced L-leucine uptake, calculated by subtracting the uptake values
obtained in water-injected oocytes from the uptake values in
poly(A) RNA-injected oocytes. Uptake of L-leucine in water-injected oocytes were 7.3 ± 0.5 and
7.4 ± 0.3 pmol/30 min/oocyte in the absence or in the presence
of L-arginine, respectively. Results were obtained with
6-8 oocytes per condition in a representative experiment. Another
independent experiment showed similar results. The antisense effect and
the L-arginine inhibition in the control and sense groups, but
not in the antisense groups, were statistically significant
(Student's t test; p
0.01).
Figure 2: Comparison of the predicted and partial amino acid sequence of OK cell rBAT protein with the human, rabbit, and rat counterparts. The first line shows the OK cells rBAT amino acid sequence. Below, only substituted amino acids in the sequences of human (second line), rabbit (third line), and rat (fourth line) rBAT proteins are shown. Amino acid gaps in the sequence are indicated by dashes. This fragment of the OK cell rBAT protein is 220 amino acid residues long and in this alignment starts at the amino acid residue 373 of the human rBAT protein. This fragment of the OK cells rBAT protein is 72%, 71%, and 70% identical (85%, 86%, and 84% similarity) to the human, rabbit, and rat rBAT protein fragment, respectively. Two putative transmembrane domains, corresponding to the second and third domains of the four-transmembrane-domain model for the rBAT protein proposed by Tate and co-workers (27) are underlined in the OK cell protein sequence. Two (OK cell protein) and three (human, rabbit, and rat proteins) potential N-glycosylation sites are boxed.
Figure 3:
Sodium-independent uptake of amino acids
in OK cells. Uptake of 50 µML-[S]cystine, L-[
H]arginine, or L-[
H]leucine by OK cells were measured
in the absence (Control) or in the presence of the indicated
unlabeled L-amino acids at 5 mM concentration in the
uptake MGA medium. Uptake values (nmol/mg protein
min) are the
mean ± S.E. from 5-12 determinations from two to four
independent experiments. All the inhibitions shown were statistically
significant (Student's t test at least p
0.01) with the exception of L-cystine transport in the
presence of L-glutamate.
The sodium-independent
uptake of L-cystine, and part of the L-arginine and L-leucine uptake, showed a pattern of substrate inhibition
very similar to the b-like transport activity
elicited by rBAT in oocytes (27) . Thus, L-cystine
uptake was almost completely abolished by a 100-fold excess of L-arginine and L-leucine in the uptake medium; in
contrast, L-glutamate did not affect L-cystine
transport, precluding expression of system X
Kinetic analysis of L-cystine uptake, examined over a range of concentration from
5 µM to 450 µM, showed apparent K and V
values of 370
± 40 µM and 13.6 ± 1.0 nmol/mg
protein
min, respectively (data not shown). In agreement with a
previous report on OK cells(31) , one high affinity system
appears to be present in OK cells. Kinetic analysis of L-arginine and L-leucine was performed over the range
of 5 µM to 1000 µM. For arginine, one single
kinetic component was observed with apparent kinetic parameters of K
211 ± 48 µM and V
59.9 ± 6.6 nmol/mg protein
min for L-arginine (data not shown). For the uptake of L-leucine inhibitable by 5 mML-arginine,
the apparent kinetic parameters were: K
175
± 56 µM and V
20.8 ±
2.5 nmol/mg protein
min (data not shown). These low apparent K
values for L-cystine, L-arginine, and L-leucine are of the same order
(slightly higher) as those reported for these amino acids via the
b
-like amino acid transport system elicited by
human, rabbit, and rat rBAT cRNA in Xenopus oocytes(1, 2, 27, 28, 40) .
Recently, it has been reported that system b-like
expressed by rBAT in oocytes shows trans-stimulation, suggesting that
this transport system is an amino acid
exchanger(3, 4, 5) . To provide further
evidence for the presence of system b
-like transport
activity in OK cells, we searched for trans-stimulation of L-arginine efflux by different amino acids. Efflux was very
low in the absence of amino acids (
1% of the total 2-min loading
of L-[
H]arginine) but was increased by
amino acid substrates of system b
-like in the
external medium: 17-fold by 1 mML-arginine, 6-fold
by 1 mML-leucine, and >2-fold by 200 µML-cystine (Fig. 4). In contrast, L-arginine efflux was not trans-stimulated by 1 mML-glutamate in the external medium (Fig. 4). The
finding that most (
70%) of the L-arginine uptake in OK
cells was due to system b
-like activity and that
y
L activity was not present in these cells indicates
that trans-stimulation of L-arginine efflux by L-leucine is due to system b
-like.
Similarly, L-leucine efflux was trans-stimulated by 1 mML-arginine (6-fold) and by 300 µML-cystine (2.5-fold) (data not shown), also showing
trans-stimulation with characteristics of system
b
-like.
Figure 4:
Trans-stimulation of arginine efflux in OK
cells. Cells were preloaded with 50 µML-[H]arginine for 2 min, then the
medium was washed out and L-[
H]arginine
efflux was measured for 45 s in media containing no amino acids (none)
or the indicated L-amino acids at 1 mM, except for L-cystine, which was present at 200 µM. Efflux
rates (mean ± S.E.) are counts/min (
1000) measured in
the medium/mg protein
min from triplicates from a representative
experiment. Efflux rates in the L-cystine, L-leucine,
and L-arginine groups were statistically different
(Student's t test; p
0.05) from those of
the none and L-glutamate groups. Another five independent
experiments gave similar results.
Finally, we investigated the polarity of
system b-like activity in OK cells grown on
transwell filters. Sodium-independent L-cystine (Fig. 5) and L-arginine (data not shown) uptake were
higher (6-8-fold) in the apical pole. This is in full agreement
with a previous report of L-cystine uptake on OK
cells(31) . Interestingly, the uptake of L-cystine and L-arginine in the apical but not in the basolateral pole
showed the characteristic inhibition pattern of system
b
-like; L-cystine uptake was abolished by a
100-fold excess of L-arginine (Fig. 5), and the uptake
of L-arginine was strongly inhibited (<80% inhibition) by a
100-fold excess of L-leucine (data not shown). In all, these
results demonstrate that OK cells express system
b
-like amino acid transport activity in the apical
pole.
Figure 5:
Polarity of the arginine-inhibitable
cystine uptake in OK cells. Uptake of 50 µML-[S]cystine by OK cells grown on
filters was measured through the apical or basolateral poles, and in
the absence (open bars) or in the presence (filled
bars) of 5 mM unlabeled L-arginine. Uptakes were
determined in the absence of sodium (MGA medium). Uptake values
(pmol/min/filter) are the mean ± S.E. from triplicates in a
representative experiment. Amino acid uptake inhibitions in the apical
pole were statistically significant (Student's t test; p
0.01). Another independent experiment showed similar
results.
Figure 6:
A,
Northern blot analysis for human rBAT-sense and antisense expression
and for the endogenous rBAT mRNA in transfected OK cells. A human rBAT
cDNA probe hybridized to a transcript of 1.2 kb in length, which
is present in the RNA from OK cells transfected with rBAT-sense or
rBAT-antisense, but absent in nontransfected cells (OK) (left panel). 30 µg of total OK cell RNA was loaded in
each lane. An OK cell rBAT cDNA probe, corresponding to the
PCR-amplified cDNA fragment shown in Fig. 2, hybridized with a
transcript of >2.3 kb present in non transfected OK cells (control)
and in rBAT-sense and rBAT-antisense OK transfected cells total RNA (middle panel). 30 µg of OK cell total RNA was loaded per
lane. The level of rBAT mRNA was reduced in the OK cells transfected
with rBAT-antisense to
20% of that in control and rBAT-sense
groups, as revealed by scanning densitometry in two independent
Northern blot analyses. Ethidium bromide staining of the Northern blot
membrane shown in the middle panel (right panel). B, amino acid uptake in OK cells transfected with human rBAT
sense or antisense. Uptake of 50 µML-[
S]cystine, L-[
H]arginine, or L-[
H]leucine by OK cells was measured in
the absence or in the presence of the indicated unlabeled L-amino acids or the amino acid analog BCH at 5 mM concentration in the uptake MGA medium. Uptake values (nmol/mg
protein
min) correspond to the uptake inhibited by the indicated
amino acids or analog and are the mean ± S.E. from triplicates
of a representative experiment. Total uptake values (i.e. in
the absence of inhibitors) in the sense and antisense groups were,
respectively: 1.50 ± 0.10 and 0.61 ± 0.03 nmol/mg
protein
min for L-cystine uptake, 10.5 ± 0.3 and
6.0 ± 0.3 nmol/mg protein
min for L-arginine
uptake, and 6.0 ± 0.5 and 4.6 ± 0.2 nmol/mg
protein
min for L-leucine uptake. All the components
shown by amino acid inhibition were statistically different from zero
value (Student's t test; at least p
0.05).
Similar results were obtained in one to three more independent
experiments.
Cell clones AS1 (antisense) and S1 (sense)
were used for further experiments in which system
b-like activity was studied. Consistently (i.e. from passage 20 to 22), b
-like transport
activity was lower (40-55%) in the AS1 antisense clone than in
the S1 sense clone (Fig. 6B). System
b
-like transport activity was almost identical in
the S1 sense clone and in control untransfected cells ( Fig. 3and 6B). The b
-like transport
activity expressed by rBAT cRNA in oocytes shows hetero-exchange
between L-arginine and L-leucine(3, 4, 5) . This has also
been suggested for this transport activity in OK cells in the present
study (Fig. 4). In order to demonstrate that rBAT is responsible
for this activity via b
-like transporter,
trans-stimulation of L-arginine efflux by L-leucine
was studied in AS1 antisense and S1 sense clones. This
trans-stimulation was reduced to
50% in the AS1 antisense clone.
Thus, efflux (cpm/mg protein
min, corrected for the radioactivity
loading after 2 min of 50 µML-[
H]arginine of 85,000 cpm/mg
protein) was 1,300 ± 1,100 and 1,200 ± 600 in the
presence of 1 mML-glutamate in the medium for S1
sense and AS1 antisense clones, respectively. This efflux was
trans-stimulated to 17,500 ± 900 and 9,200 ± 300 by 1
mML-leucine in the medium of S1 sense and AS1
antisense cells, respectively (data are mean ± S.E. of
triplicates from a representative experiment). In contrast to system
b
-like transport activity, the uptake of L-leucine inhibited by the amino acid analog BCH, model for
system L, was unaffected in the AS1 antisense clone (Fig. 6B). Similarly, the sodium-dependent uptake of 50
µML-leucine was not affected in the antisense
AS1 clone (i.e. the uptake values from triplicates of a
representative experiment were: 4.7 ± 1.4 and 4.0 ± 0.7
nmol/mg protein
min for the S1 sense and the AS1 antisense clones,
respectively). This demonstrates that rBAT antisense expression in OK
cells results in a specific decrease in system
b
-like transport activity. Kinetic analysis of L-cystine uptake showed that rBAT antisense expression reduces
the apparent V
(7.8 ± 0.8 and 3.5
± 0.7 nmol/mg protein
min, for the S1 sense and the AS1
antisense clones, respectively) without a significant effect on the
estimated apparent K
values (227 ± 39 and
285 ± 50 µM, for the S1 sense and the AS1 antisense
clones, respectively) (Fig. 7).
Figure 7:
Kinetic analysis of L-cystine
uptake in OK cells transfected with human rBAT sense or antisense.
Uptake of L-[S]cystine was measured at
varying concentrations in the absence of sodium in the uptake medium,
both in OK cells transfected with rBAT-sense (filled symbols)
or with rBAT-antisense (open symbols). Eadie-Hofstee
transformations of 5 mML-arginine-inhibitable L-cystine uptake are shown. Uptake values (nmol/mg
protein
min) are the mean ± S.E. from triplicates of a
representative experiment. When not visible, S.E. bars are smaller than
symbols.
Here we have shown that system b-like is
the major component of the transport of cystine in the apical pole of
the opossum kidney cell line OK. This system is very similar to the
exchanger activity of amino acids elicited by rBAT cRNA in oocytes. In
addition, we have demonstrated that rBAT expression is necessary for
this b
-like amino acid transport activity in this
renal epithelial cell line. Due to the role of human rBAT gene
in type I cystinuria(8, 10, 12) , a corollary
of the present study is that the b
-like amino acid
exchanger would be defective in this type of cystinuria.
Identification of the amino acid transport activity associated with
rBAT in renal cells has so far been elusive. No functional expression
of the amino acid transport activity associated with rBAT has been
obtained in mammalian cells (e.g. COS cells)(30) . All
the data previously reported on the amino acid transport activity
associated with rBAT were obtained in Xenopus oocytes
(reviewed in (41) ). In addition, the rBAT protein has a low
predicted number of transmembrane
domains(25, 27, 28) , and indirect evidence
suggested its association with an unidentified subunit of 35-50
kDa to give a putative functional complex of 125 kDa in kidney and in
oocytes expressing rBAT ((29) ). All this prompted
the hypothesis that rBAT is not an amino acid transporter by itself,
but rather full expression of the b
-like exchanger
can only be achieved by association with an endogenous subunit of the
oocyte. In this situation, description of the amino acid transport
activity associated with rBAT in renal cells was a clear prerequisite
for the full understanding of the physiopathology of rBAT. The present
study demonstrates that, as in oocytes, rBAT expression is necessary
for the b
-like transport activity present in the OK
cell line. Whether rBAT acts as a modulator or as a catalytic component
of this transport activity remains to be established through
reconstitution experiments and identification of the putative subunit
linked to rBAT.
To our knowledge the present study represents the
first description in renal epithelial cells of
b-like amino acid transport activity, defined as
high affinity sodium-independent heteroexchange diffusion for cystine,
dibasic (e.g.L-arginine) and neutral (e.g. leucine) amino acids. Only fragmental views of this amino acid
transport activity have been described in kidney and intestine. Thus,
functional studies indicated a high affinity reabsorption system for L-cystine in the proximal straight tubule of the nephron (23, 24) (i.e. S3 segment, where rBAT has
been localized; (6) and (7) ). This was also shown to
be present in the small intestinal mucosa and to be defective in
biopsies from patients with
cystinuria(20, 21, 42) . This high affinity
system is shared with dibasic amino acids and shows heteroexchange
diffusion of dibasic amino acids and
cystine(17, 18, 19, 42) . Transport
of L-cystine, examined at low (µM) concentration,
is inhibited by some L-neutral amino acids, suggesting a high
affinity cystine transporter shared with neutral amino
acids(18, 22, 23, 24) . The sodium
dependence of cystine reabsorption has been controversial for years;
the high affinity cystine uptake in rat brush border membranes was
first believed to be sodium-dependent and, later,
sodium-independent(18, 43) . After the molecular
identification of rBAT and its role in type I cystinuria, it was
necessary to characterize the amino acid transport activity associated
with rBAT in renal epithelial cells and test their consistency with the
previously reported data on renal cystine reabsorption. Among the
different renal epithelial cell lines analyzed, the high affinity
sodium-independent cystine-dibasic amino acid transport system
evidenced in renal preparations was also substantiated in OK
cells(14, 31) . Here we have shown that this transport
system is also shared by neutral amino acids, is due to rBAT
expression, and is very similar, if not identical, to the
b
-like amino acid exchanger expressed by rBAT in
oocytes.
All the foregoing suggests the following as a plausible
description of the role of rBAT in L-cystine renal
reabsorption. The sodium-independent b-like amino
acid exchanger (i.e. associated to rBAT) is responsible for L-cystine reabsorption in the brush border of the epithelial
cells of the S3 segment of the nephron; a defect in this reabsorption
system causes type I cystinuria. In contrast to the S3 segment of the
nephron, the bulk of cystine reabsorption occurs in the S1-S2 segment
of the nephron(44) , and it seems to be due to a low affinity,
high capacity system that appears to be unshared with dibasic amino
acids and which most probably is sodium-dependent and not detectable in
the small
intestine(17, 18, 19, 23, 24, 42, 45) .
Due to the genetic heterogeneity in cystinuria (11, 12) and the very mild, if any, alteration of
cystine absorption in the small intestine in type III cystinuria, the
putative low affinity, high capacity cystine reabsorption system is an
obvious candidate for this type of cystinuria.
Given that there is
no clear agreement on the sodium dependence of the renal reabsorption
of cystine, it was suggested that the main driving force for this
reabsorption may be the intracellular reduction to L-cysteine,
which then leaves the cell by a basolateral transport
system(44) . The exchange of amino acids via system
b-like in oocytes expressing
rBAT(3, 4, 5) , and also suggested for this
transport activity in OK cells by the results presented here, offers an
additional mechanism of accumulation for cystine and dibasic amino
acids in the brush border membranes of the epithelial cells of the S3
segment of the nephron; exchange with intracellular neutral amino acids
could drive the active reabsorption of cystine and dibasic amino acids.
Indeed, rBAT-specific accumulation of cystine occurs in oocytes where
reduction to cysteine is prevented by diamide.
Reabsorption
of cystine and dibasic amino acids through this exchange may be favored
by intracellular reduction of cystine to cysteine and the negative
brush border membrane potential, respectively. This is consistent with
the finding that patients with type I cystinuria have mutations in the rBAT gene, and therefore a defective b
-like
amino acid exchange activity, and show hyperexcretion of cystine and
dibasic amino acids but not neutral amino acids. This heteroexchange
mechanism of cystine and dibasic amino acid reabsorption could also
explain the enigmatic secretion of cystine exceeding the glomerular
filtration rate in dogs after lysine infusion in
vivo(46, 47) ; the high concentration of lysine
in the glomerular filtrate after infusion may block by competition the
cystine reabsorption through system b
-like in the
epithelial cells of the S3 segment and stimulate the exchange through
the brush border membrane of lysine (inward) and neutral amino acids
(outward), including cysteine, which could be oxidized to cystine in
the lumen. The final result would then be a cystine urinary excretion
higher than the cystine that reaches the kidney for glomerular
filtration.
Finally, the OK cells could be envisaged as an ideal
model with which to study the cell biology and regulation of this
transport activity associated with rBAT. The b-like
amino acid exchanger seems to be the only transport system for cystine
in the apical pole of these cells. Aside from the ontogenic regulation
of rBAT expression in kidney(6) , little is known about the
hormonal regulation of this reabsorption system for cystine and dibasic
amino acids.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X95475[GenBank].