Arginine transport through system y+L in cultured
human fibroblasts: normal phenotype of cells from LPI
subjects
Valeria
Dall'Asta1,
Ovidio
Bussolati1,
Roberto
Sala1,
Bianca Maria
Rotoli1,
Gianfranco
Sebastio2,
Maria Pia
Sperandeo2,
Generoso
Andria2, and
Gian C.
Gazzola1
1 Dipartimento di Medicina Sperimentale, Sezione di
Patologia Generale e Clinica, Plesso Biotecnologico Integrato,
Università degli Studi di Parma, 43100 Parma; and
2 Dipartimento di Pediatria, Università Federico II, 80131 Napoli, Italy
 |
ABSTRACT |
In
lysinuric protein intolerance (LPI), impaired transport of cationic
amino acids in kidney and intestine is due to mutations of the
SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino
acid (CAA) transport in human fibroblasts obtained from LPI patients
and a normal subject. In both cell types the bidirectional fluxes of
arginine are due to the additive contributions of two Na+-independent, transstimulated transport systems. One of
these mechanisms, inhibited by N-ethylmaleimide (NEM) and
sensitive to the membrane potential, is identifiable with system
y+. The NEM- and potential-insensitive component,
suppressed by L-leucine only in the presence of
Na+, is mostly due to the activity of system
y+L. The inward and outward activities of the two systems
are comparable in control and LPI fibroblasts. Both cell types express
SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as
well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We
conclude that LPI fibroblasts exhibit normal CAA transport through
system y+L, probably referable to the activity of
SLC7A6/y+LAT2.
cationic amino acid; membrane transport; system y+; membrane potential; nitric oxide; lysinuric protein intolerance
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INTRODUCTION |
LYSINURIC PROTEIN
INTOLERANCE (LPI, also known as dibasicoaminoaciduria type II;
MIM 222700) is an autosomal recessive disorder in which increased
urinary excretion of cationic amino acids (CAA) and protein intolerance
are caused by impaired CAA transport across the basolateral membrane of
kidney and intestine cells (see Ref. 20 for review). Early
works on nonepithelial cells (21, 22) suggested that human
fibroblasts (HF) but not erythrocytes from LPI patients express the
genetic defect as a reduced transstimulation of CAA efflux.
When those studies were performed, no hints were available about the
molecular bases of either LPI defect or, more in general, CAA transport
itself; moreover, a single mechanism, named system y+
(27, 28), was thought to account for CAA transport in
mammalian cells. It is now well known that, in fact, system
y+ corresponds to the activity of several carriers of the
cationic amino acid transporter (CAT) family (16, 17),
none of which, however, has been found mutated in LPI patients
(15). Moreover, detailed studies, performed in the last
few years (10) indicated that mammalian cells can
transport CAA through at least four distinct mechanisms, namely,
systems y+, y+L, b0,+, and
B0,+ (see Table 1).
Among these, system y+L, first described by Deves et al.
(11), is an exchange route that recognizes CAA in the
absence of Na+ but requires the cation to interact with
neutral amino acids such as leucine. The same authors also found that
the activities of systems y+L and y+ can be
easily discriminated by taking advantage of the sensitivity of the
latter mechanism to N-ethylmaleimide (NEM) inhibition
(9). System y+L is a glycoprotein-associated
amino acid heterodimeric transporter (18, 24, 26) whose
"heavy chain" is the glycoprotein 4F2hc, and the hydrophobic
"light chain," y+LAT1 or y+LAT2, is responsible for the recognition
and operational features of the transport process (26) .
Functional and genetic studies have suggested that system
y+L is a candidate for the transport mechanism mutated in
LPI (24). This hypothesis has been validated independently
by two groups (1, 25) that identified SLC7A7,
encoding for the light chain y+LAT1, as the gene mutated in LPI
patients. This gene is expressed not only in kidney and intestine but
also, although to a lesser degree, in nonepithelial tissues (1,
25).
In light of these recent developments, we have readdressed here
the issue of the expression of LPI defect in nonepithelial tissues. To
this aim, we have performed a characterization of bidirectional CAA
fluxes in HF and compared the discriminated activities of systems
y+ and y+L in control cells and in cells
obtained from LPI patients.
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MATERIALS AND METHODS |
Cell strains, cell culture, and experimental procedures.
Normal HF were obtained from a 15-yr-old healthy donor. LPI cells were
obtained from skin biopsies of two patients affected by LPI.
SLC7A7 cDNA of LPI1 cells presents a deletion of
543 bp at position 197, while LPI2 cells present an
insertion of 4 bp (ATCA) at position 1,625 of the SLC7A7
cDNA (1, 23). Cells were routinely grown in 10-cm-diameter
dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal bovine serum (FBS), 4 mM L-glutamine, and 5.5 mM
glucose. The conditions of culture were as follows: pH 7.4, atmosphere
5% CO2 in air, and temperature 37°C. The experiments
were made on fibroblast subcultures resulting from 3 × 104 cells seeded onto 2-cm2 wells of disposable
24-well trays (Nunc) and were incubated for 5-7 days in 1 ml of
growth medium. The culture medium was always renewed 24 h before
the experiment. Cells were used at a density of 25 ± 10 µg
protein/cm2.
For amino acid-free incubation, cell monolayers were washed twice
in Earle's balanced salt solution (EBSS) and incubated in the same
solution supplemented with 10% dialyzed FBS.
Amino acid influx.
All the experiments were performed by using the cluster tray method for
the measurement of solute fluxes in adherent cells (13)
with appropriate modifications. Cell monolayers, washed twice with
modified bicarbonate-free EBSS buffered at pH 7.4 with 20 mM
Tris · HCl, were incubated for 30 s in the same solution containing [14C]arginine. In this interval of time,
arginine uptake approached linearity (2). The experiment
was terminated by three rapid washes (<10 s) in 0.1 M
MgCl2, and cell monolayers were extracted in 0.2 ml
ethanol. The radioactivity of cell extracts was determined with
Microbeta Trilux (Wallac). Extracted cell monolayers were then
dissolved with 0.5% sodium deoxycholate in 1 M NaOH, and protein
content was determined directly in the well by using a modified Lowry
procedure as previously described (13) .
NEM inhibits selectively system y+ and, thus, can be
employed to discriminate the components of CAA uptake (9).
In cultured HF, NEM treatment incompletely suppresses arginine influx
(Fig. 1). To eliminate the contribution of
system y+, NEM was added to the incubation medium 5 min
before the uptake assay at a final concentration of 0.4 mM from a 100×
stock solution in water. Preliminary experiments had shown that, under
the conditions adopted, arginine transport was not further inhibited by
the prolongation of NEM treatment.

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Fig. 1.
L-Arginine (L-Arg) influx in
cultured human fibroblasts: sensitivity to N-ethylmaleimide
(NEM). Cells grown in DMEM supplemented with 10% fetal bovine serum
(FBS) were treated for 5 min with the indicated concentrations of NEM
and washed twice with Earle's balanced salt solution (EBSS). Arginine
uptake was then assayed with 30-s incubations in the same solution
supplemented with 20 µM [14C]arginine. Data points are
means of 3 independent determinations with SD indicated when greater
than point size. Lines represent the best fit of experimental data to a
rectangular hyperbola. Regression parameters were 48 ± 12.2 µM
for half-maximal inhibitory concentration ([I]0.5) and
57.4 ± 2.2 nmol · ml cell
water 1 · min 1 for maximal
inhibition (Imax). V, velocity of initial
L-arginine influx.
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In the experiments in which Na+-independent
transport was to be measured, NaCl and NaH2PO4
were replaced, respectively, by N-methyl-D-glucamine (NMG) and choline salts to
employ a modified Na+-free EBSS (NMG-EBSS). When the
extracellular K+ concentration
([K+]o) was to be changed, KCl replaced NaCl
in EBSS and 25 mM HEPES-NaOH or -KOH was employed as a buffer.
The osmolality of the transport solutions was routinely checked with a
vapor pressure osmometer (Wescor 5500) and was found to be 280 ± 10 mosmol/kg.
Expression of influx data.
The intracellular fluid volume was estimated by urea distribution
space, as described previously (6, 7). Amino acid influx
is expressed as micromoles or nanomoles per milliliter of intracellular
water per minute.
Kinetic parameters of arginine influx were determined by nonlinear
regression analysis by using Eq. 1 for a saturable system plus diffusion
|
(1)
|
where V is the initial velocity of amino acid influx,
Vmax is the maximal velocity of transport, [S]
is the amino acid substrate concentration, Km is
the Michaelis-Menten constant, and Kd is the
diffusion constant, and by using Eq. 2 for a competitively inhibited system
|
(2)
|
where V0 is the influx velocity in the
absence of the inhibitor, [I] is the inhibitor concentration,
Imax is the maximal inhibition, and [I]0.5 is
the inhibitor concentration required for half-maximal inhibition.
Amino acid efflux.
Determination of arginine efflux was performed by sequentially removing
aliquots of the extracellular medium and replacing them with fresh
solution as described by Rotoli et al. (19) for the efflux
of neutral amino acids. Briefly, after a 6-h depletion in EBSS
supplemented with 10% dialyzed FBS, cells were loaded for 15 min with
0.5 µCi/ml of carrier-free [14C]arginine in EBSS
(arginine concentration [Arg] = 1.5 µM). After this period, cells
were washed twice with fresh EBSS in the presence or in the absence of
Na+ and incubated in 0.4 ml of the same solution.
[14C]arginine that had escaped from cells was measured
after 30 s and 1 min, whereas the amino acid remaining in the
cells was determined after extraction of cell monolayers in 0.2 ml of
ethanol. Efflux data are expressed as percentages of the total arginine
accumulated before the efflux.
Reverse transcription.
Total RNA from subconfluent cultures of control and LPI fibroblasts,
seeded onto 10-cm2 wells, was isolated with Trizol (Life
Technologies Italia). RNA (5 µg) that was pretreated with RNase-free
DNase, heated at 70°C for 10 min, and placed on ice for 1 min was
then incubated with a mixture containing 0.5 mM dNTP mix, 25 ng/µl
oligo(dT)15-18 (Life Technologies), 10 mM
dithiothreitol, 1× first strand buffer, 10 units of RNase inhibitor
(Pharmacia), 200 units of SuperScript RT (Life Technologies), and water
to a final volume of 20 µl for 1 h at 42°C. The reaction was
stopped by heating at 70°C for 15 min. The duplex of RNA-DNA was
treated with 5 units of RNase H (USB) at 37°C for 20 min, and the
amount of single-strand cDNA was evaluated by fluorometry (Victor,
Wallac) with the fluorescent probe Oligreen (Molecular Probes) by using
phage M13+ as single-strand DNA standard.
PCR.
Single-strand cDNA (200 ng) from each sample was amplified in a total
volume of 50 µl with 1.25 units of Taq DNA polymerase (Qiagen), 1× PCR buffer, 0.2 mM each dNTP, and 1.5 mM
MgCl2 along with transporter-specific sense and antisense
primers (see Table 2) at a concentration
of 0.4 mM. The primers were designed according to the known sequences
reported in GenBank with the help of the Primer3 program (19a).
The "hot start" technique was used to increase amplification
efficiency. After an initial denaturation at 94°C for 3 min, samples
were heated to 80°C to reduce formation of nonspecific amplification
products. Taq polymerase (1.25 units) was then added, and
the reaction went on with the annealing step at 59°C for 45 s,
followed by the extension step at 72°C for 2 min. A further 34 cycles
were carried out, with the only difference being the denaturation step
of 30 s. A final extension of 5 min at 72°C was performed.
Amplified mixtures (10 µl each) were electrophoresed through a 2%
agarose gel (NuSieve 3:1-FMC), stained with ethidium bromide (1 µg/ml) in 1× TBE buffer (0.1 M Tris, 90 mM boric acid, and 1 mM
EDTA, pH 8.4), and visualized by ultraviolet light. Product size was
established by comigration with a 100-bp ladder marker (Life
Technologies Italia). Pictures of the electrophoresed cDNAs were
recorded with a digital DC 120 Kodak camera.
Materials.
FBS and culture medium (DMEM) were purchased from BioWhittaker.
[14C]arginine (312 mCi/mol) was obtained from du Pont de
Nemours (Bad-Homburg, Germany), and ethanol was from Carlo Erba (Milan, Italy). Sigma (St. Louis, MO) was the source of all other chemicals.
 |
RESULTS |
Characterization of arginine transport in cultured HF.
Little difference was observed when the kinetic analysis of arginine
influx was performed in the absence or in the presence of
Na+ (Fig. 2). The parameters
obtained (Table 3) indicate that, under both conditions, arginine influx can be described as the additive result of a single saturable system, endowed with a moderately high
affinity for the substrate (Km
60 µM), and a nonsaturable component, formally indistinguishable from
diffusion in the concentration range adopted. However, the apparent
homogeneity of the transport process, although consistent with the
linearity exhibited in the Hofstee plot (Fig. 2B), contrasts
with the incomplete inhibition by NEM, which reduced
Vmax of arginine influx by <70%. NEM-resistant arginine influx is mediated by a single saturable agency whose affinity
for arginine (Km = 45 µM) is slightly
higher than that exhibited by total influx. These results indicate that
two main components, both Na+ independent, account for CAA
transport in cultured HF.

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Fig. 2.
Kinetic analysis of L-arginine influx in
cultured human fibroblasts: Na+-independence and inhibition
by NEM. A: cells grown in DMEM supplemented with 10% FBS
were washed twice in EBSS (Na+ present) or
N-methyl-D-glucamine (NMG)-EBSS (Na+
absent), as indicated. Arginine uptake was then assayed with 30-s
incubations in the same solution supplemented with
[14C]arginine at concentrations ranging from 0.001 to 0.5 mM. Before uptake, when indicated, assay cells were treated with NEM
(0.4 mM) in the last 5 min of incubation in DMEM. Uptake was
terminated, and cells were processed as described in MATERIALS
AND METHODS. Data points are means of 3 independent
determinations with SD indicated when greater than point size. Lines
represent the best fit of experimental data to Eq. 1 (see
MATERIALS AND METHODS). Regression parameters are shown in
Table 3. B: the same influx data from A are
reported according to the Eadie-Hofstee graphical representation, after
subtraction of the nonsaturable component.
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Table 3.
Kinetic constants of L-arginine transport in the presence
and absence of sodium: effect of NEM treatment
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Figure 3 shows the effects of leucine on
arginine influx in either the presence or absence of Na+.
In NEM-treated cells (Fig. 3A), leucine inhibited arginine
influx. However, the maximal inhibition by the neutral amino acid was much larger in the presence of Na+, when >80% of the
NEM-resistant arginine influx was leucine inhibitable, than in the
absence of the cation (Imax < 20%). Leucine
inhibition was restricted to the NEM-insensitive component of arginine
influx, because the NEM-sensitive fraction was not significantly
affected by L-leucine up to a concentration of 1 mM (Fig.
3B). These results indicate that most of the
NEM-insensitive, leucine-inhibitable portion of arginine influx is due
to the activity of system y+L, while the NEM-sensitive,
leucine-resistant fraction can be identified as system y+
(10).

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Fig. 3.
L-Arginine influx in cultured human
fibroblasts: inhibitory effect of L-leucine
(L-Leu). Cells grown in DMEM plus 10% FBS were washed
twice in EBSS or Na+-free NMG-EBSS, as indicated. Arginine
uptake was then assayed with 30-s incubations in the same solution
employed for washing supplemented with 20 µM
[14C]arginine in the presence of the indicated
concentrations of L-leucine. A: NEM-resistant
fraction. Cells were pretreated with 0.4 mM NEM for the last 5 min of
incubation in DMEM. Lines represent the best fit of experimental data
to Eq. 2 (see MATERIALS AND METHODS). Regression
parameters for influx values determined in the presence of
Na+ were [I]0.5 = 355 ± 77.3 µM
and Imax = 36.55 ± 1.366 nmol · ml cell
water 1 · min 1; parameters for
influx values determined in the absence of Na+ were
[I]0.5 = 205 ± 45.3 µM and
Imax = 12.1 ± 2.17 nmol · ml cell
water 1 · min 1. Data points are
means of 3 independent determinations with SD indicated when greater
than point size. B: NEM-sensitive fraction. Data points
represent the difference between arginine influx data (means of 3 independent determinations) obtained in NEM-untreated and NEM-treated
cells at any indicated concentration of leucine.
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Operational features of systems y+
and y+L in cultured HF.
The kinetic analysis was repeated under discriminating conditions in
both fed and depleted cells to study the effect of changes in the
intracellular amino acid pool on the arginine influx pathways. The
results indicate that both systems y+ and y+L
are significantly transstimulated (Fig. 4).
The effect was much larger for system y+, whose
Vmax is more than fourfold enhanced in fibroblasts with an
intact amino acid pool compared with that in cells depleted for 6 h in an amino acid-free saline solution. The
Vmax of system y+L was only doubled
in fed cells compared with their depleted counterparts (Table
4).

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Fig. 4.
Contribution of systems y+ and
y+L to L-arginine influx in fed or depleted
cultured human fibroblasts: Eadie-Hofstee graphical representation. The
influx assay was performed as described in Fig. 1 immediately after
having been washed twice in EBSS (fed cells) or after an extensive 6-h
depletion of fibroblasts in EBSS plus 10% FBS (depleted cells).
A: system y+, in which uptake was measured in
the presence of 5 mM L-leucine. B: system
y+L, in which cells were incubated for 5 min in 0.4 mM NEM
before the uptake assay. Data points are means of 3 independent
determinations. Lines represent the saturable components of arginine
influx whose kinetic parameters are shown in Table 4.
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While the dependence on membrane potential has been conclusively
described for system y+ (14), no clear-cut
data are available about effects of voltage changes on the activity of
system y+L in nucleated cells, although in human
erythrocytes (8) and in plasma membrane vesicles from
placenta (12) this system has been described as poorly
dependent on membrane potential. In the experiment shown in Fig.
5, membrane potential of cultured human fibroblasts was changed by increasing [K+]o
from 5 to 150 mM in the presence of the K+ ionophore
valinomycin (100 µM). Under these conditions, the membrane potential
of HF is dominated by the K+ transmembrane gradient and
ranges from
4 to
72 mV (3). To reduce the possible
influence of transstimulation by the intracellular amino acid pool on
transport activities, we extensively depleted cells with a 6-h
incubation in EBSS + 10% FBS. As expected, the activity of system
y+ exhibited an evident dependence on
[K+]o. The relationship between system
y+ activity and log[K+]o is
fairly described by a linear regression (slope
11.13 ± 0.94;
P < 0.01; R2 = 0.979). On
the contrary, system y+L activity was not significantly
affected by changes in [K+]o (slope
1.199 ± 1.5; not significantly different from 0, P = 0.59). This result indicates that, in cultured HF,
system y+L activity is substantially voltage insensitive.

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Fig. 5.
Dependence of the discriminated components of arginine
influx on the extracellular K+ concentration
([K+]o). Cells were depleted for 6 h in
EBSS supplemented with 10% dialyzed FBS. After this period, cells were
washed twice with modified EBSS at different
[K+]o, as indicated, and were incubated for 3 min in the same solutions supplemented with 3% dialyzed FBS. The
influx of 20 µM L-[14C]-arginine was then
measured at different [K+]o. Valinomycin (100 µM) was added during the 3-min incubation at different
[K+]o and maintained during the influx assay.
System y+, influx assayed in the presence of 1 mM
L-leucine; system y+L, cells treated with 0.4 mM NEM in the last 5 min of incubation in EBSS plus 10% dialyzed FBS.
Data points are means of 3 independent determinations with SD indicated
when greater than point size. Lines represent linear regressions.
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Expression of genes related to CAA transport in cultured HF.
Figure 6 shows the results of RT-PCR analysis
of CAA transporters expressed in normal HF. System y+
activity corresponded to the expression of SLC7A1 (coding
for hCAT1 transporter), SLC7A2 (coding for hCAT2B and 2A
transporters), and, possibly, SLC7A4 genes (Fig.
6A). The expression of two genes for y+L-related
light chains, SLC7A6 (coding for y+LAT2) and
SLC7A7 (coding for y+LAT1), was also detected in
fibroblasts, along with a clear-cut expression of the gene
MDU1 for the corresponding heavy chain 4F2hc/CD98.

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Fig. 6.
Expression of genes involved in cationic amino acid
transport in normal fibroblasts. RT-PCR products obtained with the
primers of the indicated genes (see Table 2) are shown. In mock
reactions lacking template (see MATERIALS AND METHODS), no
detectable bands were found (not shown). The experiment was repeated
twice with comparable results.
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Comparison of discriminated systems for arginine influx in control
and LPI cells.
Figure 7 shows the results of an experiment
in which the discriminated arginine influx was measured in control
cells and in fibroblasts obtained from two LPI patients carrying
different mutations of SLC7A7 (see Cell strains, cell
culture, and experimental procedures). In both the presence and
absence of Na+, the values of total and discriminated
arginine influx determined in control fibroblasts were intermediate
between those obtained in the two LPI strains. No clear-cut
quantitative or qualitative alteration was therefore detectable in LPI
cells compared with control fibroblasts. These data, obtained in fed
cells, were confirmed in amino acid-depleted fibroblasts (data not
shown); under the latter condition, total arginine influx, as well as
its system-specific components, was lower than corresponding values
obtained in fed cells, as expected for transstimulated transport
mechanisms.

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Fig. 7.
Arginine uptake in control in control and lysinuric
protein intolerance (LPI) fibroblasts. Cells grown in DMEM supplemented
with 10% FBS were washed twice in EBSS (Na+ present) or
NMG-EBSS (Na+ absent), as indicated. Arginine uptake was
then assayed with 30-s incubations in the same solution supplemented
with 20 µM L-[14C]-arginine. A:
control cells. B: LPI1 cells. C:
LPI2 cells. +NEM, cells treated with 0.4 mM NEM in the last
5 min of incubation in DMEM plus 10% FBS; +Leu, arginine influx
assayed in the presence of 1 mM L-leucine. Points are means
of three independent determinations with SD indicated.
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Comparison of discriminated arginine efflux in control and LPI
cells.
The transport defect in LPI is thought to cause an impairment of CAA
efflux through the basolateral membrane of kidney and intestine
epithelium (26), suggesting that the malfunction of system
y+L in LPI cells is restricted to CAA efflux. To assess
this hypothesis, the contribution of systems y+ and
y+L to arginine efflux was discriminated and compared in
control (Fig. 8, A and
B) and LPI1 fibroblasts (Fig. 8, C
and D). After an extensive depletion of the intracellular
amino acid pool, cells were incubated with tracer amounts of arginine
for 15 min. In a subset of cells, NEM was added during the last 2 min
of uptake so as to block system y+ operation selectively.
NEM-treated cells exhibited a slower efflux with respect to untreated
cells in both the presence (Fig. 8, A and C) and
absence of Na+ (Fig. 8, B and D), as
expected from the inhibition of system y+. However, when
arginine efflux was performed in the presence of extracellular leucine
(100 µM), the efflux rate was significantly increased.
Leucine-induced transstimulation was observed only in the presence of
Na+, and, therefore, it is clearly attributable to the
operation of system y+L. The results obtained in the other
LPI cell strain, LPI2 fibroblasts, were similar (data not
shown).

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Fig. 8.
Arginine efflux in control and LPI fibroblasts.
After a depletion of 6-h incubation in EBSS supplemented with 10%
dialyzed FBS, cells were loaded for 15 min in the presence of 1.5 µM
L-[14C]-arginine in EBSS. NEM (0.4 mM) was
added as indicated during the last 2 min of incubation. After this
period, cells were washed in EBSS (Na+ present;
A and C) or NMG-EBSS (Na+ absent;
B and D), as indicated, and incubated for 1 min
in 0.4 ml of the same medium in the presence or absence of 100 µM
L-leucine. A and B: control cells.
C and D: LPI1 cells. Arginine efflux
was calculated as described in MATERIALS AND METHODS. Data
points are means of 3 independent determinations with SD indicated when
greater than point size.
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The behavior of the three cell strains was thus comparable not only
with respect to the overall arginine efflux (NEM absent, leucine
absent) but also to the basal (NEM treated, leucine absent) or even the
transstimulated (NEM treated, leucine present) outward activity of
system y+L.
Expression of y+L-related genes in
control and LPI fibroblasts.
The comparable phenotype of system y+L activity exhibited
by control and LPI fibroblasts prompted us to study the expression of
genes coding for y+L-related light chains in the three cell
strains employed in this study (Fig. 9). The
expression of SLC7A6, coding for y+LAT2 (24), was clearly detectable and similar in the three cell strains. On the
contrary, the expression of SLC7A7, which codes for y+LAT1 light chain (1, 18, 24, 25), was evident only in control and LPI2 fibroblasts, although it was also detectable in
LPI1 fibroblasts under more sensitive amplification
conditions (1).

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Fig. 9.
Expression of y+L-related genes in control
and LPI fibroblasts. Semiquantitative RT-PCR analysis of gene
expression was performed in the 3 cell strains by employing the primers
indicated in Table 2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
transcript was co-amplified with transporter transcripts. Amplification
started from 200 ng of cDNA. In mock reactions lacking template (see
MATERIALS AND METHODS), no detectable bands were found (not
shown). The experiment was repeated twice with comparable results.
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 |
DISCUSSION |
Arginine transport was previously characterized in cultured HF and
was found to be dependent on the activity of a single transport system,
identified as system y+ (28). At variance with
those data, the present contribution demonstrates that HF transport
arginine through two main Na+-independent routes. One of
these routes exhibits the typical features of system y+ and
is referable to the expression of several members of CAT family (cf.
Fig. 6). The other mechanism involved in arginine influx can be
identified with system y+L because of its inhibition by
leucine in the presence, but not in the absence, of Na+ and
its insensitivity to NEM treatment. The results of the kinetic analysis
presented in Table 4 indicate that the affinity of system y+L for arginine is of the same order of magnitude as that
exhibited by system y+ and, therefore, is markedly lower in
cultured HF than in red blood cells (10). The roughly
comparable affinities exhibited by systems y+ and
y+L explain why the heterogeneity of CAA transport had been
overlooked in previous works with HF.
The small inhibition of arginine influx by leucine, detectable in the
absence of Na+ (cf. Fig. 3), could point to the presence of
the so-called system b0,+ (10). However,
because this fraction accounts for at best 7-8% of the total
arginine influx at either 20 µM (cf. Fig. 3) or physiological plasma
concentrations (not shown), the contribution of transport systems other
than y+ or y+L to arginine influx appears
marginal in HF.
Previous studies on CAA transport in HF indicated that system
y+ operates as an exchange strictly dependent on the
membrane potential. Because of this characteristic, the activity of the
system, measured as either the initial influx of L-arginine
or the steady-state accumulation of the amino acid, has been employed
as a voltage indicator (2, 3). Conversely, the evidence
presented here demonstrates that, in the same cells, as in other models
(8, 12), system y+L exhibits poor, if any,
sensitivity to voltage changes. However, the discriminated contribution
of system y+ to the total influx of arginine is substantial
and can well explain why the intracellular levels of the CAA exhibit a
clear-cut voltage dependence.
The absence of voltage dependence of system y+L activity
indicates the substantial electroneutrality of the rate-limiting step of the transport process mediated by the system and points to an as yet
uncharacterized coupling of CAA influx with cation efflux or anion
influx. Because chloride substitution with impermeant organic anions
does not affect the activity of the system (8), the
electroneutrality of the transport process mediated by the system
should be referred to the efflux of a cation. On the other hand,
K+ countertransport seems unlikely because system
y+L activity is not affected by the increase of the
extracellular concentration of this cation (8, 12; present study).
Therefore, we favor the hypothesis that the system works as a
homoexchange or, alternatively, a heteroexchange, coupling CAA
influx with a combined efflux of intracellular Na+ and
neutral amino acids. In fact, Chillaron and coworkers (4) failed to detect any efflux of neutral amino acids promoted by arginine
influx in y+L-expressing oocytes. These authors, however,
demonstrated in the same model that the expression of system
y+L causes an increased transmembrane ratio of its
substrates and, as a consequence, defined the system as a tertiary
active amino acid transport system (4), although it is not
clear what kind of energy source the system employs to build up the
transmembrane substrate gradient. The definition of the bioenergetic
features of system y+L deserves, therefore, further investigations.
Arginine efflux has been characterized with an approach similar to that
employed for influx discrimination. The results presented in Fig. 8
indicate that CAA efflux is very rapid with almost 80% of the
intracellular tracer exiting cells in the first minute of the amino
acid-free incubation. NEM treatment markedly lowers arginine efflux,
while the NEM-resistant component is significantly transstimulated by
leucine in the presence, but not in the absence, of Na+.
These data indicate that CAA efflux is shared by systems
y+L and y+ in HF and point to the bidirectional
activity of both systems in mesenchymal cells.
This conclusion is of great potential interest for the elucidation of
the pathogenesis of extrarenal alterations present in LPI. Indeed,
while the main defect in this rare autosomal recessive disease is a
decreased absorption of CAA in kidney, the phenotype of LPI patients
also involves alterations in different organs, such as hemophagocytic
lymphohistiocytosis or alveolar proteinosis (20), that are
hardly accounted for by the kidney defect. In 1987 Smith et al.
(22) actually found that transstimulation of homoarginine
efflux was deficient in LPI fibroblasts and concluded that LPI defect
is expressed in mesenchymal tissues.
The recent identification of SLC7A7 as the gene mutated in
LPI (1, 25) now allows a direct reassessment of the
conclusions reached by Smith et al. (22).
SLC7A7 codes for y+LAT1, one of the proteins that associates
with 4F2hc/CD98 to yield system y+L activity (18,
24). However, at least one other protein, y+LAT2, can bind to
4F2hc to express y+L transport activity (18, 24,
26). Moreover, the different affinities exhibited by system
y+L in oocytes and erythrocytes suggest that different
members of the SLC7A family account for system
y+L in the various models (18). In this report
we have demonstrated that HF are endowed with system y+L
activity and express at least two members of the SLC7A
family related to this transport system. However, the activity of the system appears comparable in cells obtained from LPI patients or from a
normal control subject. It should be stressed that LPI cells employed
here were derived from two patients carrying different mutations. Under
the same conditions, the expression of SLC7A7 is evident in
LPI2 and control cells but not in LPI1
fibroblasts, although these cells do indeed express the gene
(1). This result could point to a lower content of
SLC7A7 mRNA in LPI1 cells, which carry a heavily
deleted gene. On the other hand, in LPI2 cells the gene has
a missense mutation, and its expression is comparable to that detected
in control cells. However, qualitative and quantitative analysis of
bidirectional arginine transport yields comparable results in the two
LPI strains. Because all the cell strains clearly express
SLC7A6, encoding for y+LAT2, it is likely that the activity of system y+L in HF is accounted for by this member of
SLC7A family. Alternatively, it is possible that both y+LAT1
and y+LAT2 are functional in normal HF. In this case y+LAT2 activity
would somehow compensate for the absence of functional y+LAT1 in LPI
cells. The elucidation of possible differential expression of y+LAT2
protein in LPI and control fibroblasts requires the availability of
specific antibodies for the transporter.
In conclusion, LPI-associated mutations of SLC7A7/y+LAT1 do
not cause any significant functional alteration in CAA transport in HF.
Consequently, other cell models should be employed to define the
metabolic role of y+LAT1 in nonepithelial cells.
 |
ACKNOWLEDGEMENTS |
This study was partially supported by Ministero dell'
Università e della Ricerca Scientifica e Tecnologica Integrated
Project "Metabolic and Molecular Bases of Inherited Diseases" (V. Dall'Asta and G. Sebastio), Consiglio Nazionale delle Ricerche Target
Project "Biotechnology" (Rome, Italy) (V. Dall'Asta), and
Telethon-Italy Grants E.652 (to G. Sebastio) and 29cp (to M. P. Sperandeo). M. P. Sperandeo is an Assistant Telethon Scientist.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: V. Dall'Asta, Dipartimento di Medicina Sperimentale, Sezione di Patologia Generale e Clinica, Plesso Biotecnologico Integrato, Università degli Studi di Parma, Via Volturno 39, 43100 Parma, Italy (E-mail: valeria.dallasta{at}unipr.it).
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. Section 1734 solely to indicate this fact.
Received 5 April 2000; accepted in final form 28 June 2000.
 |
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