Department of Cell and Molecular Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Normal lung function
requires transepithelial clearance of luminal proteins; however, little
is known about the molecular mechanisms of protein transport. Protein
degradation followed by transport of peptides and amino acids may play
an important role in this process. We previously cloned and
functionally characterized the neutral and cationic amino acid
transporter ATB0+ and showed expression in the lung by mRNA
analysis. In this study, the tissue distribution, subcellular
localization, and function of the transporter in native tissue were
investigated. Western blots showed expression of the ATB0+
protein in mouse lung, stomach, colon, testis, blastocysts, and human
lung. Immunohistochemistry revealed that ATB0+ is
predominantly expressed on the apical membrane of ciliated epithelial
cells throughout mouse airways from trachea to bronchioles and in
alveolar type I cells. Electrical measurements from mouse trachea
preparations showed Na+- and Cl-dependent,
amino acid-induced short-circuit current consistent with the properties
of ATB0+. We hypothesize that, by removing amino acids from
the airway lumen, the transporter contributes to protein clearance and,
by maintaining a low nutrient environment, plays a role in lung defense.
mucociliary clearance; airway surface liquid; acute respiratory distress syndrome; pulmonary alveolar phospholipoproteinosis; glucose transport
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INTRODUCTION |
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THROUGHOUT THE LUNG, proteins and peptides such as mucins, lysozyme, transferrin, defensins, and surfactants are secreted in the airway lumen and alveolar space. The architecture of the lung requires that proteins must be removed from the airway surface (27, 48). The mucociliary escalator transports a portion of the protein up the airways, but most of the protein must eventually traverse the epithelium (9). Several studies have shown that exogenously applied proteins can cross the lung epithelium by endocytosis and paracellular diffusion (6-9, 16, 23, 24, 29, 33); however, the mechanisms of transepithelial transport of endogenous proteins are not known. Transepithelial protein transport from the airway surface may also be mediated by protein degradation followed by transport of peptides and amino acids. Proteases and peptidases are expressed in the lung (4, 26, 28, 40, 44, 51). Although their role in protein degradation has not been studied, amino acid concentrations have been measured in the airway surface liquid (ASL; see Refs. 15 and 41). The presence of proteases and amino acids in the airway lumen suggests that protein degradation and removal of the degradation products by specific transporters is an important mechanism of protein clearance from the airway and alveolar space.
Our laboratory cloned and functionally characterized the human
amino acid transporter B0+ (hATB0+, SLC6A14),
which is a Na+- and Cl-dependent broad (B)
specificity transporter for neutral (0) and cationic (+)
amino acids (B0+; see Ref. 39). The highest
hATB0+ mRNA levels were detected in the lung and trachea
(39). Previous work demonstrated amino acid transport with
characteristics similar to that of ATB0+ at the apical
membrane of human bronchial and rat alveolar epithelial cells in
culture (10, 22). Therefore, we hypothesized that ATB0+ mediates transport of amino acids from lumen of the
airway and alveolar space. In this report, we used antibodies raised
against human and mouse ATB0+ to determine the tissue
distribution and subcellular localization of the transporter. In
addition, measurements of amino acid-induced short-circuit current
(ISC) were performed on excised mouse tracheas mounted in Ussing chambers. These studies suggest that the transporter is expressed on the luminal membrane of the airway and alveolar epithelium. By removing amino acids from the lung lumen,
ATB0+ may contribute to protein clearance and, by
maintaining a low nutrient environment, play a role in lung defense.
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MATERIALS AND METHODS |
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Plasmids.
The coding region of hATB0+ (GenBank accession no.
NM_007231, nucleotides 74-2027) was amplified by PCR and ligated
into the EcoR I sites of pcDNA3.1 (Invitrogen, Carlsbad,
CA). The coding region of mATB0+ (GenBank Accession no.
AF161714, nucleotides 247-2229) was amplified by PCR and ligated
into pcDNA3.1/CT-GFP-TOPO, but the protein was not fused to the GFP tag
(Invitrogen). These constructs will be referred to as
pcDNA-hATB0+ and pcDNA-mATB0+, respectively.
Cell culture. HEK293 (ATCC CRL-1573) and COS-7 (ATCC CRL-1651) cells were maintained in DMEM containing 4,500 mg/l glucose, 10% FBS, and 100 units penicillin/streptomycin and split every 5-7 days using trypsin-EDTA (GIBCO, Carlsbad, CA). HEK293 or COS-7 cells were seeded in 100-mm dishes (1.5 × 106 cells/dish) for membrane preparation. Transfections were performed the following day using Effectene according to the manufacturer's instructions (Qiagen, Valencia, CA) and harvested at 40-48 h posttransfection.
Membrane preparation. HEK293 or COS-7 cells transfected with pcDNA-hATB0+ or pcDNA-mATB0+ were washed with PBS and pelleted by centrifugation at 700 g for 5 min. Hypotonic lysis buffer (20 mM HEPES, pH 7.5, 20 mM NaCl, and 5 mM EDTA containing protease inhibitors 10 µM leupeptin, 10 µM pepstatin A, 4 µM aprotinin, and 30 µg/ml phenylmethylsulfonyl fluoride) was added to the cell pellet. Cells were lysed by four freeze-thaw cycles, and lysate was centrifuged for 5 min at 700 g to remove nuclei and cellular debris. The supernatant was subsequently centrifuged at 100,000 g for 20 min at 4°C in a Beckman TLA 100.2 rotor to pellet the membrane fraction. The high-speed pellet was resuspended in the same lysis buffer containing 1% Triton X-100 to solubilize membrane proteins.
Human lung samples were obtained from Scott Randell at the University of North Carolina (UNC) Cystic Fibrosis Pulmonary Research and Treatment Center. The human lung samples originated from patients with varying pathology: emphysema (patient 1); cystic fibrosis (patients 2 and 3); normal (patients 4 and 5). The cartilaginous airways collected were ~1-2 cm in diameter and are designated by the patient number followed by "a" for airway. Distal lung samples were obtained from the most distal 1-2 cm of the human lung and are designated by the patient number followed by "dl" for distal lung. All tissue samples were rinsed in ice-cold PBS, immediately frozen in liquid nitrogen, and stored atAntibodies.
Peptides corresponding to a region of the NH2-terminus of
mouse ATB0+ (CGENDENQERGNWSKKSDY), COOH-terminus of human
ATB0+ (CADHEIPTVSGSRKPE), and the COOH-terminus of mouse
ATB0+ (CEKHRGERYRDMAEPAKETDHEI) were synthesized and
conjugated to keyhole limpet hemocyanin by the UNC Microprotein
Sequencing and Peptide Synthesis Facility and used to raise rabbit
polyclonal antibodies (Covance, Princeton, NJ). Antibodies were tested
by Western blot analysis for their ability to detect the
ATB0+ proteins from transfected cell lysate and to
recognize COOH- or NH2-terminal
glutathione-S-transferase (GST) fusion proteins (data not
shown). NH2-terminal mATB0+ antibodies did not
detect recombinant protein and thus were not effective for Western
blotting (data not shown). The COOH-terminal hATB0+
antibodies (-hATB0+) were effective in recognizing the
recombinant protein in transfected cells and hATB0+
COOH-terminal GST fusion proteins. Also, the COOH-terminal
mATB0+ antibodies (
-mATB0+) were effective
in recognizing the recombinant protein in transfected cells and
mATB0+ COOH-terminal GST fusion proteins. For Western
blots,
-hATB0+ was affinity purified using the
immunizing peptide conjugated to an UltraLink Iodoacetyl column
(Pierce, Rockford, IL). For mouse lung immunostaining,
-mATB0+ was purified over a protein G column, MAbTRAP,
according to the manufacturer's instructions (Amersham Biosciences).
Anti-
-tubulin IV was used for colocalization studies in mouse lung
(Biogenex, San Ramon, CA). Mouse monoclonal anti-actin antibodies were
used as a control in tissue Western blots (Chemicon, Temecula, CA).
SDS-PAGE and Western blots. Protein samples in Laemmli sample buffer were separated by 8% SDS-PAGE and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was washed in blocking solution, 5% dehydrated milk in Tris-buffered saline containing Tween 20 (5 mM Tris, pH 7.5, 7.5 mM NaCl, and 1% Tween 20) for 30 min at room temperature, and then incubated in primary antibody in blocking solution for 1 h at room temperature or 4°C overnight. The secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), was added to the blot at a 1:10,000 dilution for 1 h. SuperSignal West Pico Chemiluminescent Substrate was used to visualize proteins recognized by antibodies (Pierce). Digital images were captured with Image Station 440 (Kodak, New York, NY).
Immunohistochemistry.
COS-7 cells were plated on coverslips coated with fibronectin (Sigma,
St. Louis, MO) and transfected with pcDNA-hATB0+ or
pcDNA-mATB0+ as described. At 24-48 h, cells were
fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with
0.5% Triton X-100 in PBS for 20 min, and incubated in blocking
solution (5% goat serum in PBS) for 1 h at room temperature.
Cells were incubated with -hATB0+ or
-mATB0+ at a dilution of 1:1,000 in blocking solution at
4°C overnight and the following day exposed to secondary antibody
(Alexa Fluor 488 conjugated goat anti-rabbit IgG) for 1 h
(Molecular Probes, Eugene, OR). Preimmune serum and antibodies blocked
with 20 µg/ml of the antigenic peptide for 1 h were used as controls.
Ussing chamber experiments.
Ussing chamber experiments were conducted as described previously
(13, 14). In short, adult 12-wk-old C57B6 mice were killed
by carbon dioxide exposure. Tracheas were excised and mounted on Ussing
chambers with a surface area of 0.025 cm2. Tissue was
bathed in Krebs-Ringer-bicarbonate (KRB) buffer containing (in mM) 140 Na+, 120 Cl, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO
-free experiments, sodium gluconate and
MgSO4 replaced the Cl
salts. Mannitol and
glucose (5 mM) were added to the apical and basolateral chambers,
respectively. Indomethacin (1 µM) was added bilaterally at the
beginning of each experiment to reduce spontaneous Cl
secretion. Tissues were gassed with 95% O2-5%
CO2 and maintained at 37°C during the experiments.
Amiloride (100 µM), leucine (100 µM), arginine (500 µM),
glutamate (100 µM), glucose (5 mM), and UTP (1 µM) were applied
sequentially to the apical chamber, and the change in
ISC was recorded.
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RESULTS |
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To gain insight into the physiological role of ATB0+
in the lung, we examined the transporter tissue distribution and
subcellular localization with antibodies generated against both human
and mouse ATB0+. Antibody characterization was performed by
Western blot analysis of membrane fractions of COS-7 or HEK293 cells
expressing pcDNA-hATB0+ or pcDNA-mATB0+.
Western blot analysis showed that -hATB0+ and
-mATB0+ recognized broad bands, ~75-85 kDa, in
membrane fractions of transfected HEK293 cells, respectively, which
were not detected in the soluble fraction (Fig.
1A), or untransfected HEK293
cells (data not shown). For both antibodies, no bands were detected in
the 75- to 85-kDa range with preimmune serum or when antibodies were
preincubated with the immunizing peptide (data not shown). The actual
molecular mass, ~75-85 kDa, is larger than the predicted molecular mass, ~72 kDa, for both human and mouse ATB0+.
There are eight and seven putative extracellular
N-glycosylation sites in the primary amino acid sequence of
human ATB0+ and mouse ATB0+ (39),
respectively, that may contribute to the difference between the
predicted and actual molecular mass and to the difference in size
between species. Antibodies were also tested for the ability to detect
hATB0+ and mATB0+ by immunocytochemistry in
COS-7 cells overexpressing the transporters. After respective antibody
incubation, immunoreactivity was detected with Alexa Flour
488-conjugated goat anti-rabbit IgG. Both
-hATB0+ and
-mATB0+ showed distinct staining of transfected cells
but not untransfected cells (Fig. 1, B and C).
Preimmune serum and antibodies preadsorbed to the immunizing peptide
showed no immunoreactivity (data not shown). Antibody characterization
demonstrated that the antibodies were specific and effective for both
Western blotting and immunostaining of ATB0+-overexpressing
cells; therefore, tissue distribution and immunolocalization experiments were subsequently initiated.
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mRNA analysis showed that hATB0+ is expressed at high
levels in the lung, fetal lung, trachea, mammary gland, and salivary
gland and at lower levels in the pituitary gland, stomach, colon,
uterus, prostate, and testis (39). To determine if the
mATB0+ protein had a similar distribution, we performed
Western blot analysis on several mouse tissues. Membrane fractions were
prepared from mouse lung, pancreas, small intestine, colon, and testis, and Western blots were probed with -mATB0+. A broad band
of ~75-85 kDa, consistent with the size of the recombinant
protein, appeared in membrane preparations of mouse lung, stomach,
colon, and testis but not in small intestine or pancreas (Fig.
2). mATB0+ protein expression
was evident throughout all regions of mouse lung: trachea, bronchi, and
distal lung (Fig. 2). Importantly, the mATB0+ protein was
also expressed early in development in mouse blastocysts (Fig. 2) where
the B0+ amino acid transport system was first described
(45).
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To determine if the human lung also expressed the ATB0+
protein, human lung samples of varying pathology were obtained from the
UNC Cystic Fibrosis Pulmonary Research and Treatment Center. Membrane
fractions of two different regions of human lung, cartilaginous airway
and distal lung, were tested for hATB0+ expression by
Western blot. An ~75- to 85-kDa band, similar in molecular mass to
the recombinant hATB0+ protein, was observed in all human
airway and distal lung protein samples (Fig.
3). Interestingly, there was a consistent
difference in protein migration on SDS-PAGE between hATB0+
protein from airway (Fig. 3, samples 1a, 2a,
4a, and 5a) and distal lung (Fig. 3,
samples 1dl, 2dl, 3dl, and
4dl). The distal lung hATB0+ appeared as a
broader band that extended into a higher molecular mass (Fig. 3). The
apparent difference in hATB0+ protein size might be
attributed to alternative splicing or posttranslational modification.
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Northern (39) and Western blot analysis (Figs. 2 and 3)
demonstrated that ATB0+ is expressed throughout the lung.
Measurements of amino acid-induced current in cultured lung epithelial
cells (10, 22) suggest the presence of an amino acid
transporter with properties similar to that of ATB0+ at the
apical membrane (39). Experiments were initiated to determine the cellular and subcellular localization of
ATB0+ in mouse lung. Immunohistochemistry was performed on
10 µm frozen sections of inflation-fixed mouse trachea and whole lung
using protein G-purified -mATB0+ (Figs.
4-7).
Immunolocalization revealed mATB0+ expression in the
epithelial cells of the trachea, bronchioles and alveoli (Figs.
4-7), and bronchi (data not shown) of mouse lung. Sections
incubated with
-mATB0+ preadsorbed with the immunizing
peptide were not labeled (Fig. 4B). These data indicate that
immunolocalization was specific for ATB0+ (Fig. 4). In both
upper and lower airways, immunohistochemistry revealed that
mATB0+ is expressed in the epithelial cells lining
the trachea (Fig. 5B), bronchi (data not shown), and
bronchioles (Figs. 4 and 6B). To determine the specific cell
type expression of mATB0+, an antibody against
-tubulin
IV, a protein enriched in cilia, was used as a marker for ciliated
cells (Figs. 5A and 6A; see Ref.
42). mATB0+ colocalized with
-tubulin IV in
ciliated columnar epithelial cells of the trachea (Fig. 5C)
and ciliated cuboidal cells of the bronchioles (Fig. 6C). In
the bronchioles, nonciliated cells showed a low level of immunostaining
(Fig. 6B). These data suggest that ATB0+ is
expressed at higher levels in ciliated cells of the airways but may
also express in the secretory Clara cells lining the lower airways
(Fig. 6). Colocalization with
-tubulin IV also suggests that
ATB0+ is expressed at the luminal side of epithelial cells
lining the airways (Figs. 5 and 6). In addition to the airways,
immunostaining of mouse lung using
-mATB0+ also showed
expression of the transporter in the alveolar epithelium (Figs. 4 and
7). There was a continuous strong network of staining throughout the
distal lung, and costaining with nuclear marker, TO-PRO3, revealed that
the majority of cells in the distal lung expressed mATB0+
(Fig. 7). Because alveolar type I cells comprise 95% of the alveolar surface area, these data indicate that ATB0+ is expressed
in alveolar type I pneumocytes (Fig. 7A). Because of the
squamous nature of alveolar type I cells, it was difficult to determine
the subcellular distribution; however, mATB0+ appears to be
localized to the membrane of alveolar type I cells (Fig.
7B). Because alveolar type II cells only comprise 5% of the
alveolar surface area, it was more difficult to determine if these
cells expressed mATB0+. However, based on the morphology
and localization of type II cells in a cornering pattern in the
alveolar sac, we concluded that type II cells might also express
mATB0+ (Fig. 7B).
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The amino acid transporter ATB0+ has been functionally
characterized in heterologous expression systems (17, 39,
43), and these studies have determined that neutral and cationic
amino acids are transported in a Na+- and
Cl-dependent manner. Because of its ion-coupled transport
mechanism, the process is electrogenic in nature and can be measured
using electrophysiological recordings. We and others have demonstrated that application of amino acids induced an inward current in
Xenopus oocytes expressing the transporter (39,
43). To detect ATB0+ function in native tissue, we
measured ISC on excised mouse tracheae mounted
in Ussing chambers. These experiments were conducted in KRB
containing NaCl or with Na+-free buffer in which
Na+ was replaced with NMDG or Cl
-free buffer
in which gluconate was substituted for Cl
. Amino acids
and glucose were applied sequentially to the apical surface of the
mouse trachea, and changes in ISC were
quantified. In the presence of amiloride, the first application of
arginine (500 µM) and leucine (100 µM) generated changes in
ISC current that were 2.06 ± 0.37 µA/cm2 (n = 14) and 2.72 ± 0.35 µA/cm2 (n = 4), respectively (Table
1 and Fig.
8). In contrast, glutamate (100 µM)
did not generate current (Fig. 8A). If arginine and
leucine induced current by different mechanisms, subsequent application of the other amino acid should result in an additive current. We
observed that application of leucine after arginine and vice versa
resulted in no additional current (Fig. 8), suggesting the amino acids
share a common mechanism and are likely to be carried by the same
ion-coupled transporter protein. The properties of the amino
acid-induced
ISC are in agreement with the
known function of ATB0+ as a neutral and cationic but not
anionic amino acid transporter.
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Another property of ATB0+ is that amino acid uptake and
transport current depend on the presence of extracellular
Na+ and Cl (39). In solution
lacking Na+, arginine failed to generate a change in (
)
ISC (Table 1). However, we observed a response
to 1 µM UTP, which stimulates Cl
secretion
(14), suggesting that the preparations were viable (data
not shown). Arginine also failed to generate current under Cl
-free conditions (Table 1), but amiloride-sensitive
current was present (data not shown). The Na+ and
Cl
dependence of the arginine-induced
ISC is consistent with the characteristics of
ATB0+ and is likely to represent ATB0+
function, specifically since ATB0+ is the only known
Na+- and Cl
- dependent cationic (e.g.,
arginine) transporter.
Transporters for other organic molecules may also be localized to the
apical membrane of the airway epithelium. In addition to arginine- and
leucine-induced current, we observed a current associated with
application of 5 mM glucose to the apical chamber, which was 2.79 ± 0.31 µA/cm2 (Fig. 8 and Table 1). This current was
present after application of leucine and arginine, indicating that it
is mediated by an independent transporter (Fig. 8). The glucose
response was virtually absent (0.13 ± 0.08 µA/cm2)
in preparations lacking Na+; therefore, glucose is likely
to be transported by a Na+-coupled transporter (Table 1).
However, under Cl-free conditions, the glucose-induced
current was similar in magnitude to the glucose response in KRB, in
accordance with the properties of known Na+-coupled glucose
transporters (Table 1 and Ref. 50). In most cases,
application of phlorizin (1 mM), an inhibitor of the
Na+-coupled glucose transporter, SGLT1 (18),
reversed the glucose-induced
ISC. These data
suggest that a Na+-coupled glucose transporter of the
SGLT-SLCA5 cotransporter family is localized at the apical membrane of
epithelial cells in the mouse trachea.
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DISCUSSION |
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The goal of this study was to gain an understanding of the
physiological roles of ATB0+ by investigating the tissue
distribution, subcellular localization, and function in mouse and human
tissues. Antibodies created against both human and mouse
ATB0+ were specific and effective for recognizing the
recombinant proteins on Western blots (Fig. 1A) and in
transfected cells by immunostaining (Fig. 1, B and
C). After initial characterization of the antibodies, the
tissue distribution of the ATB0+ proteins was explored. The
mATB0+ protein was detected by -mATB0+ on a
Western blot in mouse stomach, colon, testis, and blastocysts and
throughout the lung (Fig. 2). The tissue distribution of the ATB0+ protein correlates with functional measurements in
various cells and tissues (Figs. 2 and 3). Na+-dependent
neutral and cationic amino acid transport, designated system
B0+, was described in mouse blastocysts (45),
Xenopus oocytes (31), a human colon cell line
(5), rabbit conjunctiva (20, 21), rabbit
small intestine (34, 35), rat anterior pituitary cultures (46), bullfrog lung (30), and human bronchial
(10) and rat alveolar (22) epithelial cells
in culture. Studies of ATB0+ mRNA expression in human
(39) and mouse (17, 43) are also in agreement
with the distribution of the protein. hATB0+ mRNA analysis
showed high expression in the trachea, lung, fetal lung, mammary gland,
and salivary gland and lower levels in the pituitary, stomach, colon,
uterus, prostate, and testis (39). mATB0+ mRNA
was detected by Northern blot in mouse lung, distal small intestine,
cecum, and colon (17, 43). In contrast, mATB0+
mRNA levels were present at low levels in the small intestine (17); however, the protein was not detected in our
preparation (Fig. 2). Further investigation is required to determine if
the ATB0+ protein is expressed in all cells and tissues in
which the mRNA was detected.
Similar to the mouse lung (Fig. 2), human cartilaginous airways and distal lung, comprised mostly of alveoli, showed expression of the ATB0+ protein (Fig. 3). Interestingly, we observed that the ATB0+ protein obtained from human airway and human distal lung showed an apparent difference in molecular mass (Fig. 3). Similarly, ATB0+ from mouse blastocysts and testis also showed different migration on SDS-PAGE compared with ATB0+ from mouse lung (Fig. 2). The variation in the apparent molecular mass might be a result of alteration in the protein primary structure or differential posttranslational modification. Because of the single size of ATB0+ mRNA (39, 43), it is likely that the difference in mobility of the transporter protein is because of alteration in posttranslational modification such as the glycosylation state. It will be important to directly determine the biological significance of the different ATB0+ isoforms.
To investigate the subcellular localization of the mATB0+
protein, immunohistochemistry was performed on mouse trachea and lung sections. This study revealed that mATB0+ is expressed
throughout the mouse lung in the epithelial cells of the trachea,
bronchioles and alveoli (Fig. 4-7), and bronchi (data not shown).
mATB0+ was predominantly expressed on ciliated epithelial
cells of the trachea and bronchioles and at lower levels on nonciliated
secretory Clara cells in the bronchioles (Figs. 5 and 6).
Colocalization with -tubulin IV, a cilia-enriched protein, indicated
apical localization (Figs. 5 and 6). We were unable to determine if
ATB0+ was localized to the cilia, microvilli, or apical
membrane; however, it was clear that ATB0+ was expressed on
the luminal side of epithelial cells lining the airways.
mATB0+ immunostaining overlapped with
-tubulin IV
staining, indicating that a portion of the transporter pool may reside
in the cilia (Figs. 5C and 6C). We also observed
intracellular punctate staining, which suggests that a portion of the
protein resides in intracellular membrane compartments (Fig.
6D). These intracellular pools might allow fast modulation
of the number of transporters at the cell membrane (36b). In
the alveoli, mATB0+ appeared to be primarily localized to
type I pneumocytes but may also be expressed in surfactant-secreting
alveolar type II cells (Fig. 7). Based on our immunolocalization data
and functional measurements in alveolar epithelial cells in culture
(22), ATB0+ is also likely to express in the
apical membrane of alveolar epithelial cells in vivo.
Functional measurements of hATB0+ and mATB0+ in
Xenopus oocytes (39, 43) and mammalian cells
(17) have demonstrated Na+- and
Cl-dependent neutral and cationic amino acid transport as
the hallmark of ATB0+ activity. Transporter function can be
determined directly by uptake of radiolabeled amino acids or by
measuring the transport-associated current electrophysiologically. In
Xenopus oocytes expressing human or mouse ATB0+,
amino acids generated an inward current resulting from the electrogenic nature of the transport process (36, 39). To demonstrate
that ATB0+ is functional in native tissue, we conducted
measurements of ISC in freshly excised mouse
tracheas mounted in Ussing chambers. We showed that, upon initial
application of the amino acids (arginine and leucine), an
amiloride-insensitive ISC was generated (Table 1
and Fig. 8). We applied a concentration severalfold higher than the
EC50 values of arginine and leucine so that each amino acid
could generate maximal transport current and, thus, consecutive application of the other amino acid would not generate additional current (Fig. 8). As predicted, the arginine- and leucine-induced current was not additive, suggesting that they both act via a common
mechanism. Transport of arginine and leucine by ATB0+ is
Na+- and Cl
-dependent in heterologous
systems. Similarly, the arginine-induced ISC
depends on the presence of both ions (Table 1). Importantly, ATB0+ is the only known Na+-dependent cationic
(e.g., arginine) amino acid transporter. Together these findings
strongly suggest that the current is mediated by ATB0+.
Therefore, our ISC measurements in excised mouse
tracheas are the first studies to definitively demonstrate the activity
of ATB0+ in native tissue. Furthermore, these data confirm
the immunolocalization results and suggest that the transporter is
functional at the apical membrane of the airway epithelium. Although
the physiological function of ATB0+ is not known, the
expression of ATB0+ throughout the airways and alveoli
suggests an important role in lung function.
Physiological significance of ATB0+ in the lung. Expression of an ion-coupled, high-affinity, broad-specificity amino acid transporter throughout the lumen of the airways and alveoli raises two interesting questions. First, what is the source of amino acids? Second, what is the functional significance of their removal under normal and pathophysiological conditions? Little is known about the amino acid composition of the fluid that lines the alveolar lumen and airway surface (15, 41), and the source of the amino acids in the airway lumen has not been studied. Amino acids may be secreted by the epithelium, filtrated from the pulmonary vasculature, or result from degradation of proteins in the lung lumen. From alveoli to trachea, there is a significant decrease in the surface area requiring a reduction in ASL volume and its contents of more than 4,000-fold. To account for the relatively constant height of the ASL and similar longitudinal transport rate (27, 48), the total volume of ASL and its protein contents must decrease from lower to upper airway. Consequently, the airway epithelium must remove proteins and/or their degradation products (2, 27).
There are several possible mechanisms of transepithelial protein transport as follows: 1) paracellular diffusion, 2) endocytosis/transcytosis, and 3) absorption of protein degradation products, small peptides, and amino acids via ion-coupled transporters (Fig. 9). In airway epithelial cells in culture, albumin was transported in an apical-to-basolateral direction by nonspecific protein transcytosis (7, 24). In alveolar preparations, several studies that utilized exogenously applied proteins showed that large proteins, such as albumin, transferrin, and IgG, are cleared by specific processes, for example, receptor-mediated endocytosis (6, 16, 23, 29, 33), and smaller proteins, such as granulocyte macrophage colony-stimulating factor and cytochrome C, are removed from the alveolar space by paracellular diffusion (6, 8, 9, 29, 33). Although transcytosis and paracellular diffusion were determined to be important for removal of exogenously applied protein in epithelial cultures, the fate of endogenous protein in the lung has not been studied. There is some evidence of protein degradation in airway epithelial cells (24), and several proteases and peptidases are expressed in the lung (4, 26, 28, 40, 44, 51). Therefore, we propose that protein degradation followed by transporter-mediated removal of the degradation products is a major pathway for protein removal from the ASL and alveolar space. In this study, we determined that the broad-specificity neutral and cationic amino acid transporter ATB0+ is predominantly localized to the apical membrane of ciliated epithelial cells lining the airways and in alveolar type I cells (Fig. 4-7). We also showed that the transporter is functional in native mouse trachea (Fig. 8 and Table 1). Therefore, ATB0+ may be involved in the removal and recycling of amino acids from the ASL and alveolar space. In addition, the H+-coupled peptide transporter, PEPT2, is localized at the apical membrane of airway epithelial cells (11, 12), and it is also likely to contribute to the removal of protein degradation products. The model in Fig. 9 demonstrates the possible protein removal pathways and specifically shows that ion-coupled transporters, such as ATB0+ and PEPT2, might play central roles in this process. In addition to amino acids and peptides, transporters for mono- and oligosaccharides might mediate the removal of sugar moieties of degraded glycoproteins such as mucin. In the present study, we showed that glucose also generated an amiloride-insensitive, Na+-dependent change in ISC (Fig. 8 and Table 1). Similar currents were observed in horse trachea (25).
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ACKNOWLEDGEMENTS |
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We thank Ann Sutherland at the University of Virginia and Scott Randell at the University of North Carolina Cystic Fibrosis Pulmonary Research and Treatment Center for providing mouse blastocyst and human lung samples, respectively, and Kirk McNaughton for assistance with the histological preparations. We acknowledge Sharon Milgram and C. William Davis for helpful discussions about the manuscript.
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FOOTNOTES |
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This work was supported in part by the National Alliance for Research on Schizophrenia and Depression Young Investigator Award and National Insitiute on Drug Abuse Grant P01-DA-12408.
Address for reprint requests and other correspondence: S. Mager, Dept. of Cell and Molecular Physiology, Univ. of North Carolina at Chapel Hill, CB# 7545, Chapel Hill, NC 27599 (E-mail: sela_mager{at}med.unc.edu).
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.
August 23, 2002;10.1152/ajplung.00164.2002
Received 24 May 2002; accepted in final form 2 August 2002.
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