Expression of the amino acid transporter ATB0+ in lung: possible role in luminal protein removal

Jennifer L. Sloan, Barbara R. Grubb, and Sela Mager

Department of Cell and Molecular Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 at -80°C. Mouse organs and human lung were pulverized into fine powder with a mortar and pestle. Tissues were homogenized with an electronic homogenizer (Omni International, Warrenton, VA) for 15 s in hypotonic lysis buffer. Membrane preparation of tissues was processed as described for HEK293 cells. The blastocysts were provided by Ann Sutherland at the University of Virginia and were harvested as previously described (32). Approximately 1 µg of blastocyst protein was lysed directly in sample buffer. The protein concentrations of all cell lysate and tissue samples were determined by bicinchoninic acid protein assay according to the manufacturer's instructions (Pierce).

Antibodies. 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 (alpha -hATB0+) were effective in recognizing the recombinant protein in transfected cells and hATB0+ COOH-terminal GST fusion proteins. Also, the COOH-terminal mATB0+ antibodies (alpha -mATB0+) were effective in recognizing the recombinant protein in transfected cells and mATB0+ COOH-terminal GST fusion proteins. For Western blots, alpha -hATB0+ was affinity purified using the immunizing peptide conjugated to an UltraLink Iodoacetyl column (Pierce, Rockford, IL). For mouse lung immunostaining, alpha -mATB0+ was purified over a protein G column, MAbTRAP, according to the manufacturer's instructions (Amersham Biosciences). Anti-beta -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 alpha -hATB0+ or alpha -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.

Mouse trachea and lungs were removed from C57B6 mice and inflation fixed by perfusing the trachea with a 1:1 ratio of optimum-cutting temperature (OCT) compound and PBS. Blocks of tissue were frozen in OCT and stored at -80°C. Mouse lung and trachea specimens were cryostat sectioned at a thickness of 10 µm and placed on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Slides were thawed to room temperature and immediately fixed with 2% paraformaldehyde for 20 min. Tissue was permeabilized with 0.5% Triton X-100 in PBS for 20 min. Subsequently, slides were blocked with 5% goat serum in PBS for 1 h. Primary antibodies, protein G column-purified rabbit alpha -mATB0+ and mouse anti-beta -tubulin IV (Biogenex), were incubated with the tissue in blocking solution at dilutions of 1:5,000 and 1:1,000, respectively, at 4°C overnight. In addition, for peptide-blocking experiments, alpha -mATB0+ was preincubated with 20 µg/ml of the immunizing peptide for 1 h at room temperature. Secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG, were diluted to 2 µg/ml in blocking solution, and slides were incubated for 1 h at room temperature (Molecular Probes). To stain nuclei, slides were incubated in TO-PRO3 at a 1:1,000 dilution in PBS for 30 min (Molecular Probes). Images were captured using a Zeiss fluorescent microscope with a ×10 objective using MetaMorph software and a Leica TCS-NT confocal microscope with ×40 and ×100 objectives.

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<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 0.4 H2PO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In Na+-free experiments, the Na+ in NaCl and NaHCO3 was replaced with N-methyl-D-glucamine (NMDG). In Cl--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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -hATB0+ and alpha -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 alpha -hATB0+ and alpha -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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Fig. 1.   Antibody characterization. A: particulate (part) and soluble (sol) fractions (50 µg) of human amino acid transporter B0+ (hATB0+)- and mouse ATB0+ (mATB0+)-expressing HEK293 cells were separated on an 8% SDS-PAGE gel, and the subsequent Western blot was probed with alpha -hATB0+ and alpha -mATB0+, respectively. Both human and mouse ATB0+ proteins were detected at ~75-85 kDa in the particulate fraction. COS-7 cells were transfected with pcDNA-hATB0+ (B) or pcDNA-mATB0+ (C) and immunostained with alpha -hATB0+ or alpha -mATB0+, respectively. Alexa Flour 488-conjugated goat anti-rabbit IgG secondary antibody was used to reveal ATB0+ staining. Images were captured with a Leica TCS-NT confocal microscope using a ×40 objective. Scale bar = 25 µm.

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 alpha -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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Fig. 2.   Tissue distribution of mouse ATB0+. Particulate (part or not labeled) and soluble (sol) fractions (100 µg) of mouse tissues were separated by 8% SDS-PAGE, and subsequent Western blots were probed with alpha -mATB0+. Western blots showed that the ~75- to 85-kDa mATB0+ protein was expressed in mouse trachea, bronchi, distal lung, whole lung, stomach, colon, testis, and blastocysts but not in small intestine or pancreas. Blots were then stripped and reprobed with alpha -actin.

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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Fig. 3.   ATB0+ in human lung. Human lung samples from five patients (1-5) of varying pathology were obtained from the University of North Carolina Cystic Fibrosis Pulmonary Research and Treatment Center. a, Cartilaginous airway samples; dl, distal lung samples. Membrane fractions (100 µg) were separated by 8% SDS-PAGE and Western blots probed with alpha -hATB0+. The hATB0+ protein expressed in all human lung samples but showed an apparent difference in molecular mass in airway samples compared with distal lung samples. The Western blot was stripped and reprobed with alpha -actin.

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 alpha -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 alpha -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 beta -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 beta -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 beta -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 alpha -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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Fig. 4.   Immunostaining of ATB0+ in mouse lung. Inflated mouse lung 10-µm frozen sections were immunostained with protein G column-purified alpha -mATB0+ (A) or alpha -mATB0+ that had been preincubated with the immunizing peptide (B). Alexa Flour 488 goat anti-rabbit IgG secondary antibody was used to visualize mATB0+ staining. Representative micrographs of a mouse bronchiole and alveoli indicate that the alpha -mATB0+ immunostaining was specific. This staining pattern was not apparent in sections incubated with antibodies blocked with the antigenic peptide (B). Images were captured with a Zeiss fluorescent microscope equipped with a ×10 objective. Scale bar = 100 µm.



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Fig. 5.   ATB0+ localization in mouse trachea. Mouse trachea 10-µm frozen sections were costained with rabbit alpha -mATB0+ and mouse alpha -beta -tubulin IV. Secondary antibodies, goat anti-rabbit IgG Alexa Fluor 488 and goat anti-mouse IgG Alexa Fluor 594, were used to visualize beta -tubulin IV (red) and ATB0+ (green). To stain nuclei, sections were incubated with TO-PRO3 (blue). Images were captured with a Leica TCS-NT confocal microscope using a ×40 objective. A: beta -tubulin IV was localized to the cilia at the apical membrane of tracheal epithelial cells. B: mATB0+ was localized to the apical membrane of tracheal epithelial cells. C: mATB0+ and beta -tubulin IV were colocalized at the apical membrane of tracheal epithelial cells. Scale bar = 15 µm.



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Fig. 6.   ATB0+ localization in mouse bronchioles. Mouse inflated lung 10-µm frozen sections were costained with rabbit alpha -mATB0+ and mouse alpha -beta -tubulin IV. Secondary antibodies, goat anti-rabbit IgG Alexa Fluor 488 and goat anti-mouse IgG Alexa Fluor 594, were used to visualize beta -tubulin IV (red) and ATB0+ (green). Nuclei were labeled with TO-PRO3 (blue). Images were captured with a Leica TCS-NT confocal microscope using a ×40 objective. A: beta -tubulin IV immunostaining of a mouse bronchiole showed staining localized to the cilia. B: mATB0+ was localized to the apical membrane of ciliated epithelial cells of the bronchiole. C: mATB0+ and beta -tubulin IV are colocalized at the apical membrane of bronchiole epithelium. Scale bar = 15 µm. D: higher magnification shows that mATB0+ was predominantly expressed in ciliated cells where it colocalized with beta -tubulin IV. Scale bar = 4 µm.



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Fig. 7.   ATB0+ localization in mouse alveoli. Mouse inflated lung 10-µm frozen sections were stained with rabbit alpha -mATB0+. Secondary antibody, goat anti-rabbit IgG Alexa Fluor 488, was used to visualize the ATB0+ protein (green). Nuclei were stained using TO-PRO3 (blue). Images were captured with a Leica TCS-NT confocal microscope using a ×40 objective. A: immunostaining revealed that mATB0+ was expressed in alveolar epithelial cells. Scale bar = 15 µm. B: higher magnification showing that mATB0+ was predominantly expressed on the membrane of alveolar type I cells and possibly in alveolar type II cells (arrow). Scale bar = 5 µm.

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 Delta 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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Table 1.   Changes in short-circuit current



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Fig. 8.   Short-circuit current (ISC) measurements in excised mouse trachea. Mouse tracheas were excised and mounted in Ussing chambers, and ISC was measured. After initial application of amiloride (100 µM), there was no response to application of 100 µM glutamate (A). The first application of either 100 µM leucine (A) or 500 µM arginine (B) generated a change in ISC. For both experiments, subsequent application of the other amino acid did not result in a response; however, application of 5 mM glucose did induce a change in ISC.

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 (Delta ) 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 Delta 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 Delta 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 beta -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 beta -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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Fig. 9.   Model of protein and amino acid removal in the lung. Throughout the lung, there is constant secretion of protein from secretory glands and surface epithelial cells, and, to prevent accumulation, these proteins must be removed. Although a portion of the luminal protein is transported cephalad, most of the luminal proteins should traverse the epithelium. There are several possible mechanisms of protein removal. Proteins may cross the epithelium by endoyctosis or the paracellular pathway. Alternatively, proteins may be degraded by proteases and peptidases into peptides and amino acids. Ion-coupled transporters, such as ATB0+ and PEPT2, which are expressed at the apical membrane of epithelial cells, might absorb these degradation products. Similarly, sugar transporters such as a sodium-coupled glucose transporter may contribute to the removal saccharide moieties on glycoproteins. Subsequently, facilitators and exchangers for organic molecules may transport amino acids and sugars into the bloodstream.

Ion-coupled transporters may also play an essential role during bacterial infection. Nutrients such as sugars and amino acids are required for bacterial colonization and growth. Thus the removal of these nutrients from the ASL and alveolar space may be critical for maintenance of a low nutrient environment and consequently prevention of bacterial growth. Interestingly, in cystic fibrosis patients, an increase in ASL amino acid concentration correlated with severity of respiratory disease (1, 41). Although the cause of this increase in amino acid concentration is unknown, it may contribute to sustained bacterial infection. We therefore can speculate that ATB0+ and other nutrient transporters are critical for lung defense.

Amino acid removal might be particularly crucial when proteins accumulate in the airway or alveolar space. Acute respiratory distress syndrome (ARDS) is characterized by damage of the alveolar-capillary barrier resulting in pulmonary edema where immune cells, inflammatory molecules, and protein-rich fluid enter the alveolar space and can result in alveolar cell death, inhibited respiratory function, and respiratory failure (47, 49). Pulmonary alveolar phospholipoproteinosis (PAP) is a rare pulmonary disease in which surfactant homeostasis is dysregulated, resulting in surfactant protein accumulation in the alveolar space and inhibited gas exchange (3, 37). For the resolution of these diseases, removal of protein from the alveolar space is necessary. Under these inflammatory conditions where immune cells infiltrate the alveoli and proteases accumulate (4, 19, 38), protein degradation and absorption might be even more critical for lung protein clearance. In pathological conditions such as ARDS and PAP, transporters such as ATB0+ and PEPT2 may be important for disease resolution. Interestingly, mutations in the amino acid transporter y+LAT1 result in lysinuric protein intolerance, a human disease characterized by hyperammonemia, hepatosplenomegaly, osteoporosis, and alveolar proteinosis (36a). The distribution of y+LAT1 in the lung has not been studied, but this phenotype suggests that amino acid transporters play important roles in protein removal from the alveolar space. Future studies may determine the role ATB0+ and transporters for other organic molecules in normal lung function and under pathophysiological conditions.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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