SPECIAL TOPIC
Alveolar Epithelial Ion and Fluid Transport
Na transport proteins are expressed by rat alveolar epithelial type I cells

Zea Borok, Janice M. Liebler, Richard L. Lubman, Martha J. Foster, Beiyun Zhou, Xian Li, Stephanie M. Zabski, Kwang-Jin Kim, and Edward D. Crandall

Will Rogers Institute Pulmonary Research Center and Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the alpha -subunit of the rat (r) epithelial Na channel (alpha -ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump alpha 1-subunit in AEC was assessed in situ. Na pump subunit isoform and alpha -rENaC expression was also evaluated by RT-PCR in highly purified (~95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-alpha 1 and anti-beta 1 Na pump subunit and anti-alpha -rENaC antibodies was detected, while reactivity with anti-alpha 2 Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-alpha 1 Na pump subunit antibody. Na pump alpha 1- and beta 1- (but not alpha 2-) subunits and alpha -rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.

alveolar epithelium; sodium pumps; ion transport; immunofluorescence; sodium channel


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ALVEOLAR EPITHELIUM IS comprised of two morphologically distinct cell types, cuboidal type II (AT2) cells and expansive type I (AT1) cells with long cytoplasmic processes that cover most of the internal surface area of the lung (11, 18). Active Na transport across the alveolar epithelium is believed to generate the osmotic gradient that drives alveolar fluid clearance under both normal and pathological conditions. Studies in isolated perfused lung (2, 17), alveolar epithelial cell monolayers (10), intact animals (43), and human lungs ex vivo (42) demonstrated that transalveolar fluid transport is inhibitable by both amiloride and ouabain, indicating that the predominant cellular pathways for vectorial Na clearance are apical amiloride-sensitive Na channels and basolateral ouabain-sensitive Na pumps (28, 49). Consistent with these findings, specific Na transporters have been localized to AT2 cells both in situ and in vitro. mRNA for all three subunits of the rat epithelial Na channel, alpha -, beta -, and gamma -rENaC, have been identified in freshly isolated AT2 cells (32) and in AT2 cells in adult rat lung (16, 27). In addition, several isoforms of the alpha - and beta -subunits of Na-K-ATPase have been identified in freshly isolated AT2 cells (29, 33, 41, 48) and in AT2 cells in situ (19, 45).

In contrast to AT2 cells, which have been extensively investigated with regard to their roles in both surfactant production and ion transport in adult lung, the properties of AT1 cells are less well characterized. This has been due, in large part, to difficulty in isolating highly purified populations of AT1 cells and subsequently identifying them by other than morphological means following isolation (1, 36, 53). Because AT2 cells acquire the morphological appearance and phenotypic characteristics of AT1 cells over time, AT2 cells in culture for several days have been used as a model of the alveolar epithelium with which to study AT1 cell-like properties (9, 13). With the use of this model, it has been demonstrated that alveolar epithelial cells (AEC), after several days in culture, exhibit active Na transport, which is inhibitable by both ouabain and amiloride (10). AEC also express abundant amounts of mRNA for all three Na channel subunits and alpha 1- and beta 1-Na pump subunit mRNA and protein (3, 12). Given the extensive surface area of the lung that is covered by AT1 cells, results of these studies tend to support a role for AT1 cells in transalveolar Na transport. However, specific Na transporters have not been successfully localized to AT1 cells in situ, and the role of AT1 cells in transepithelial Na transport remains presumptive.

We utilized colocalization immunofluorescence (IF) techniques to evaluate expression of Na pump and Na channel subunits in isolated AT1 cells within partially purified populations of AEC and in whole lung. Isolated AT1 cells were first identified by their reactivity with AT1 cell-specific antibodies [VIIIB2 and anti-aquaporin (AQP)-5] and their lack of reactivity with AT2 cell [surfactant protein A (SP-A)] or leukocyte [leukocyte common antigen (LCA)] markers. The alpha 1-, alpha 2-, and beta 1-subunits of Na-K-ATPase and the alpha -subunit of the rENaC (alpha -rENaC) were then localized by concurrent labeling with either VIIIB2 or anti-AQP-5. In whole lung, expression of the alpha 1-subunit in AT1 cells was assessed by concurrent labeling with anti-AQP-5. Expression of Na pump subunit isoforms and alpha -rENaC was also evaluated in highly purified (~95%) AT1 cell preparations by reverse transcriptase-polymerase chain reaction (RT-PCR). The results of these studies demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation and enrichment. AT1 cells were isolated using several modifications of previously described methods for AT2 and AT1 cell isolation (4, 5, 14, 15). Briefly, lungs from adult male Sprague-Dawley rats (250-300 g) were perfused via the pulmonary artery with RPMI 1640 containing 25 mM HEPES (solution A). Lungs were lavaged with phosphate-buffered saline (PBS; pH 7.2) containing 5 mM each of EDTA and EGTA and then filled with 10 ml of solution B (solution A with 10% dextran) containing elastase at 4.5 U/ml (Worthington, Freehold, NJ) at 37°C for a total of 40 min. Lung tissue was dissected away from the trachea and large airways and chopped in solution B containing 20% fetal bovine serum (FBS) and DNase (2 mg/ml). Lung fragments were agitated by end-over-end rotation and filtered sequentially through 100- and 20-µm Nitex mesh (Tetko, Elmsford, NY). Cells were resuspended in medium followed by panning on IgG-coated bacteriological plates for 30 min. Cytocentrifuged preparations of these partially purified AEC were processed for IF following fixation in 100% methanol for 10 min.

To further enrich for AT1 cells, partially purified cell suspensions were subjected to density gradient separation using a discontinuous iodixanol (OptiPrep; Nycomed Pharma, Oslo, Norway) gradient consisting of 2 ml each of Optiprep solutions of densities 1.068, 1.047, 1.033, and 1.012. Cells were centrifuged on the gradient at 600 g for 15 min. Cells between densities of 1.012 and 1.047 were collected, washed in Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture in a 1:1 ratio with 1% FBS, and resuspended in PBS-0.1% bovine serum albumin (BSA) at a concentration of 107 cells/ml. Immunoselection for AT1 cells was undertaken by incubating cells with the AT1 cell-specific monoclonal antibody (MAb) VIIIB2 for 30 min at 4°C. After being washed in RPMI with 1% FBS, cells were incubated with magnetic beads coated with a human anti-mouse IgG (CELLection Pan Mouse IgG kit; Dynal, Lake Success, NY) for 15 min at 4°C. Bound AT1 cells were selected by magnetic separation and resuspended in RPMI with 1% FBS. Beads were removed by incubating cells with DNase releasing buffer (Dynal) before processing for RT-PCR. Cell viability (>90%) was determined by trypan blue dye exclusion. Cell purity was assessed by IF using the AT1 cell-specific antibodies VIIIB2 or anti-AQP-5. All media and other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated and were of the highest commercial quality available.

Cell-specific antibodies. VIIIB2 is a murine MAb that was generated in this laboratory that recognizes an epitope in the apical membrane of AT1 cells in situ that is not detectable in other adult rat lung cells or other tissues (13). By IF, VIIIB2 also labels AEC in primary culture that have undergone transdifferentiation toward the AT1 cell phenotype. The polyclonal rabbit anti-AQP-5 antibody (Alpha Diagnostic, San Antonio, TX) recognizes a 28-kDa protein present in AT1 cells. This antibody has been used for both immunofluorescent labeling (6) and immunoblotting (7) to detect AQP-5 in tissues and cells, respectively, and yields equivalent results to polyclonal antisera obtained from Landon S. King (Johns Hopkins Univ.) and Chemicon (Temecula, CA) that were raised to the same peptide. Polyclonal guinea pig or rabbit anti-SP-A antibodies [J. Whitsett (31) and J. Shannon (23, 46), University of Cincinnati] were used to identify AT2 cells. Although known to be expressed in some cell types other than AT2 cells (e.g., Clara cells), SP-A is not expressed by AT1 cells. Leukocytes were identified using a murine monoclonal anti-LCA antibody (Chemicon).

Na pump and Na channel antibodies. The alpha 1-subunit isoform of Na-K-ATPase was detected using a murine MAb (6H or 464.4) obtained from M. Caplan, Yale University (24), although it is now also commercially available from UBI (Lake Saranac, NY). This antibody has been used by many investigators for detection of Na pumps in a wide variety of mammalian cells and tissues by immunohistochemical and immunofluorescent techniques (37) and can also detect Na pumps by immunoblotting (50). We and others have previously used this reagent to detect and quantify Na pump alpha 1-subunits in AEC by IF and Western blotting (3, 8, 12, 35, 55). The beta 1-subunit isoform of Na-K-ATPase was detected using a murine MAb (IEC 1/48) obtained from A. Quaroni, Cornell University (26). We have used it to identify Na pump beta 1-subunits in lung and AEC (12, 55). As described by Zhang et al. (55), it can be used to immunoprecipitate Na pump beta 1-subunits from rat lung and other tissues. The presence of the alpha 2-subunit isoform of Na-K-ATPase was evaluated using a mouse MAb (McB2) obtained from K. Sweadner, Harvard University, and has also been used widely for detection of Na pumps in many different cells and tissues by immunoblotting and immunohistochemistry (47, 50, 51). The alpha -subunit of rENaC was detected using a rabbit polyclonal antibody (1190-2) obtained from D. Benos, University of Alabama. It is an anti-alpha -bENaC (bovine ENaC) antibody that recognizes alpha -rENaC as well as alpha -bENaC but reacts negligibly with beta - and gamma -rENaC and has been used to detect cells expressing alpha -rENaC by ELISA, Western analysis (20), and IF (22).

IF (isolated cells). Fixed cytocentrifuged cell preparations were rinsed in PBS and treated with PBS-3% BSA overnight to block nonspecific reactivity. Cells were identified by double labeling with combinations of 1° antibodies specific for AT1 cells (VIIIB2 and anti-AQP-5), AT2 cells (anti-SP-A), or leukocytes (anti-LCA). In this set of experiments, specific combinations of 1° MAb and polyclonal antibodies (PAb) used to minimize cross-reactivity of 2° antibodies were VIIIB2 (MAb) and anti-AQP-5 (PAb), VIIIB2 (MAb) and anti-SPA (PAb), and anti-AQP-5 (PAb) and anti-LCA (MAb). Localization of the Na pump and Na channel subunits to AT1 cells was determined by concurrent labeling of cells with either VIIIB2 or anti-AQP-5 (used as the AT1 cell marker) and antibodies to the alpha 1-, alpha 2-, or beta 1-subunits of Na-K-ATPase or the alpha -subunit of rENaC. For the reasons noted above to avoid cross-reactivity of 2° antibodies, specific combinations of 1° antibodies used for this set of experiments were: anti-AQP-5 (PAb) and anti-alpha 1 (MAb), anti-AQP-5 (PAb) and anti-beta 1 (MAb), anti-AQP-5 (PAb) and anti-alpha 2 (MAb), and VIIIB2 (MAb) and anti-alpha -rENaC (PAb). Negative controls included substitution of normal rabbit or guinea pig serum or rabbit IgG for the PAbs, or a nonreacting mouse MAb MF20 (D. Fischman, Cornell Univ.), which is reactive only with chicken myosin for the MAbs (13). After being incubated with 1° antibodies or negative controls, slides were washed with PBS, followed by incubation with appropriate combinations of anti-mouse and anti-rabbit or anti-guinea pig 2° antibodies linked to either rhodamine isothiocyanate (red) or fluorescein isothiocyanate (FITC, green). Slides were treated with Vectashield (Vector Laboratories, Burlingame, CA) antifade mounting media and were viewed with an Olympus BX60 microscope equipped with epifluorescence optics.

IF (intact lung). Normal rat lung was inflated and fixed in 4% paraformaldehyde. After being embedded in paraffin, 2- to 5-µm sections were cut. After being deparaffinized and rehydrated through graded alcohols, slides underwent microwave antigen retrieval (Antigen Unmasking Solution; Vector) and were double labeled using the anti-alpha 1-subunit of Na-K-ATPase MAb and anti-AQP-5 PAb. Rabbit polyclonal anti-AQP-5 antibody was amplified using an avidin-biotin system with FITC (Vector), and following an avidin-biotin block step, mouse monoclonal anti-alpha 1-antibody was amplified using an avidin-biotin system with Texas red (Vector). Negative controls included substitution of normal rabbit serum or rabbit IgG for the anti-AQP-5 PAb and MF20 or MOPC21 (Sigma), an irrelevant mouse IgG1, for the anti-alpha 1-subunit antibody. Tissue sections were treated with Vectashield antifade mounting media. Images were captured using a cooled charge-coupled device camera (Magnafire; Olympus, Melville, NY) with a barrier filter equipped for simultaneous detection of Texas red and FITC. Images were imported into Adobe Phostoshop (Adobe Systems, Mountain View, CA) as TIFF files and printed in CMYK color using a Hewlett-Packard Deskjet 970C.

RNA extraction and RT-PCR. Total cellular RNA was extracted from highly purified (~95%) AT1 cell preparations immediately following cell isolation using an RNeasy mini-kit for RNA extraction from small numbers of cells (Qiagen, Valencia, CA). DNA was removed by on-column DNase treatment. RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (SuperScript II; Invitrogen Life Technologies, Carlsbad, CA) in the presence of random primers, followed by amplification with Taq polymerase using subunit-specific oligonucleotide primers for the alpha 1-, alpha 2-, and beta 1-subunit isoforms of Na-K-ATPase and the alpha -subunit of rENaC. Specific PCR primer pairs used for amplification were 1) alpha 1-subunit: 5'-TCC-TCC-CTC-TTT-CCT-CCG-3' (bases 99-116) and 5'-GCC-TCG-GCT-CAA-ATC-TGT-TC-3' (complementary to bases 423-404); 2) beta 1-subunit: 5'-CAT-CTG-GAA-CTC-GGA-GAA-GAA-GG-3' (bases 504 to 526) and 5'-TTG-GGG-TCA-TTA-GGA-CGG-AAG-3' (complementary to bases 746 to 726); 3) alpha 2-subunit: 5'-TGG-GGGTGG-CAA-GAA-GAA-AC-3' (bases 152 to 171) and 5'-CAT-AGG-CTA-AGA-AGC-AGA-GAA-GCG-3' (complementary to bases 438 to 415); and 4) alpha -rENaC: 5'-TCA-CTT-CAG-CAC-ATC-TTC-CCC-AGC-G-3' (bases 2211 to 2235) and 5'-GTA-CCT-GCC-TAC-CCG-TCC-CAA-GTG-G-3' (complementary to bases 3062 to 3038). Samples were amplified for 27-30 cycles, and PCR products were separated by agarose gel electrophoresis. Assuming that all the contaminating cells were AT2 cells, parallel experiments were performed using RNA from the number of AT2 cells equal to the number of contaminating cells present in the AT1 cell preparations, followed by PCR amplification within the linear range (27-30 cycles). Ethidium bromide-stained gels were visualized with ultraviolet light.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of isolated alveolar cells. Labeling of cytocentrifuged preparations using combinations of 1° antibodies specific for AT1 cells (VIIIB2), AT2 cells (anti-SP-A antibody), or leukocytes (anti-LCA antibody) indicates that the partially purified alveolar cell populations are comprised of approximately equal numbers of these three cell types. Immunomagnetic selection to further enrich for AT1 cells yielded ~500,000 AT1 cells per rat, with purity ranging from 70-98% and >90% viability. Preparations of ~95% purity were used for RT-PCR analysis.

Identification of AT1 cells by double labeling IF. Cytocentrifuged cell preparations were concurrently labeled with VIIIB2 and anti-AQP-5. As shown in Fig. 1, all cells that are reactive with VIIIB2 are also reactive with anti-AQP-5. Furthermore, all cells not reactive with VIIIB2 are also not reactive with anti-AQP-5. In contrast, cells reactive with VIIIB2 do not label with anti-SP-A antibody (which identifies AT2, but not AT1, cells in whole lung in situ) and vice versa (Fig. 2). Similarly, cells that are reactive with anti-AQP-5 are also not labeled with anti-SP-A, and double labeling with anti-LCA and either anti-AQP-5 or anti-SP-A indicates that neither AT1 nor AT2 cells are reactive with anti-LCA (data not shown). No reactivity was observed when normal rabbit or guinea pig serum (or IgG) was substituted for the polyclonal anti-AQP-5 and anti-SP-A antibodies, respectively, or when MF20 was substituted for VIIIB2 or anti-LCA antibodies. Colocalization of both AT1 cell markers to the same cells, together with the absence of labeling of these same cells with either anti-SP-A or anti-LCA, confirms that these AT1 cell-specific antibodies can be used successfully to identify AT1 cells within partially purified populations of alveolar cells.


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Fig. 1.   Colocalization of alveolar type I (AT1) cell-specific markers. Partially purified alveolar cells were concurrently labeled with primary antibodies specific for type I cells in situ. Phase (A) and immunofluorescence (IF) images demonstrate that all cells that are reactive with mouse monoclonal antibody (MAb) VIIIB2 (B) are also labeled with polyclonal anti-aquaporin (AQP)-5 antibody (C). Original magnification ×400.



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Fig. 2.   Localization of AT1 and alveolar type II (AT2) cell-specific markers. Isolated alveolar cells were concurrently labeled with antibodies that recognize either type I (VIIIB2) or type II [anti-surfactant protein A (SP-A)] cells in situ. Phase (A) and IF images indicate that AT1 cells (B) do not label with anti-SP-A (C). Original magnification ×400.

Identification of Na pump subunits by IF in isolated alveolar cells. Localization of the alpha 1-, beta 1-, and alpha 2-subunits was performed by concurrent labeling of cells with the AT1 cell-specific anti-AQP-5 PAb and the Na pump subunit isoform MAbs. As shown in Fig. 3, AT1 cells, identified by reactivity with anti-AQP-5, also label strongly with 6H, an antibody specific for the alpha 1-subunit of Na-K-ATPase. Similarly, AT1 cells are strongly reactive with IEC 1/48 antibody to the beta 1-subunit of Na-K-ATPase (Fig. 4). Other cells in the field (presumably AT2 cells) are also labeled with the anti-alpha 1 and anti-beta 1 Na pump subunit antibodies, but not with anti-AQP-5. No reactivity was observed when normal rabbit serum (or IgG) or MF20 were substituted for either the polyclonal rabbit anti-AQP-5 or the monoclonal anti-Na pump subunit antibodies, respectively. Neither AT1 cells nor any other cells are labeled with the anti-alpha 2 Na pump subunit antibody McB2 (Fig. 5). Rat brain, used as a positive control for alpha 2, was strongly reactive with the anti-alpha 2-subunit antibody. Double labeling with anti-SP-A and Na pump subunit antibodies confirms that AT2 cells within these partially purified populations also express the alpha 1 and beta 1 Na pump subunit isoforms (data not shown).


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Fig. 3.   Expression of alpha 1-subunit of Na-K-ATPase in isolated alveolar cells. Cells were concurrently labeled with AT1 cell-specific polyclonal anti-AQP-5 antibody and MAb anti-alpha 1 Na-K-ATPase subunit antibody 6H. Phase (A) and IF images demonstrate that AT1 cells labeled with anti-AQP-5 (B) are reactive with anti-Na pump alpha 1-isoform antibody (C). Other cells in the field, likely AT2 cells, are also reactive with the anti-alpha 1-subunit antibody. Original magnification ×400.



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Fig. 4.   Expression of beta 1-subunit of Na-K-ATPase in isolated alveolar cells. Cells were concurrently labeled with AT1 cell-specific polyclonal anti-AQP-5 antibody and MAb anti-beta 1 Na-K-ATPase subunit antibody IEC 1/48. Phase (A) and IF images demonstrate that AT1 cells labeled with anti-AQP-5 (B) react strongly with anti-Na pump beta 1-isoform antibody (C). Other cells in the field, likely AT2 cells, are also reactive with the anti-beta 1-subunit antibody. Original magnification ×400.



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Fig. 5.   Lack of expression of alpha 2-subunit of Na-K-ATPase in isolated alveolar cells. Cells were concurrently labeled with AT1 cell-specific polyclonal anti-AQP-5 antibody and MAb anti-alpha 2 Na-K-ATPase subunit antibody McB2. Phase (A) and IF images demonstrate that AT1 cells labeled with anti-AQP-5 (B) are not reactive with anti-Na pump alpha 2-isoform antibody (C). Other cells in the field, likely AT2 cells, are also not reactive with the anti-alpha 2-subunit antibody. Original magnification ×400.

Identification of rENaC alpha -subunit by IF in isolated alveolar cells. Localization of the alpha -subunit of rENaC was performed by concurrent labeling with the AT1 cell-specific MAb VIIIB2 and the anti-alpha -rENaC subunit PAb. As shown in Fig. 6, AT1 cells, identified by their reactivity with VIIIB2, are also reactive with anti-alpha -rENaC. Other cells in the field not reactive with VIIIB2 (presumably AT2 cells) are also reactive with anti-alpha -rENaC. No reactivity was observed when MF20 or normal rabbit serum (or IgG) was substituted for either the monoclonal VIIIB2 or polyclonal rabbit anti-alpha -rENaC antibodies, respectively.


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Fig. 6.   Expression of alpha -subunit of the rat epithelial Na channel (rENaC) in isolated alveolar cells. Cells were concurrently labeled with AT1 cell-specific MAb VIIIB2 and a polyclonal antibody that recognizes the alpha -subunit of rENaC. Phase (A) and IF images demonstrate that AT1 cells labeled with VIIIB2 (B) are reactive with anti-alpha -rENaC antibody (C). Other cells in the field, likely AT2 cells, are also reactive with the anti-alpha -rENaC antibody. Original magnification ×400.

Identification of alpha 1 Na pump subunit in AT1 cells in whole lung. Sections of whole rat lung were double labeled with anti-AQP-5 PAb and mouse anti-alpha 1 Na pump subunit MAb. AT1 cells are identified by labeling of the apical cell surface with anti-AQP-5 (green) (Fig. 7). As shown in Fig. 7A, linear staining (red) localizes the alpha 1-subunit of Na-K-ATPase to the basolateral surface of AQP-5-expressing AT1 cells. Substitution of mouse IgG for the anti-alpha 1 MAb reveals no reactivity with AT1 cells (Fig. 7B). Figure 7C demonstrates that relative signal intensity for the alpha 1-subunit is much greater on the basolateral membrane of AQP-5-negative AT2 cells than AT1 cells, perhaps accounting for the inability to detect AT1 cell Na pumps in situ in previous reports.


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Fig. 7.   Expression of alpha 1-subunit of Na-K-ATPase in AT1 cells in situ. Sections of rat lung tissue were incubated with polyclonal anti-AQP-5 antibody and either mouse MAb anti-alpha 1-subunit or nonspecific mouse monoclonal IgG. Labeling was detected using fluorescein isothiocyanate for AQP-5 or Texas red for alpha 1. A: linear staining (red) localizes the alpha 1-subunit of Na-K-ATPase to the basolateral surface of AT1 cells that are labeled with anti-AQP-5 antibody (green) on the apical surface. Magnification ×100. B: substitution of mouse monoclonal IgG for anti-alpha 1 antibody reveals no reactivity with AT1 cells labeled with anti-AQP-5 antibody on the apical surface. Magnification ×100. C: AT1 cells labeled on the apical surface with anti-AQP-5 antibody (green) and on the basolateral surface with anti-alpha 1 antibody (red). The AT2 cell, which is not reactive with anti-AQP-5 antibody, reacts far more intensely on the basolateral surface than adjacent AT1 cells. An autofluorescent erythrocyte appears yellow-red. Original magnification ×200.

Identification of Na pump and Na channel subunits by RT-PCR in isolated AT1 cells. RT-PCR of highly purified AT1 cell RNA demonstrates appropriately sized bands for the alpha 1- and beta 1-subunits of Na-K- ATPase and the alpha -subunit of rENaC (Fig. 8). Within the linear range of amplification, a relatively faint band is seen using RNA from a number of AT2 cells equal to the number of contaminating cells in the AT1 cell preparations (Fig. 8). Consistent with the IF data reported above in isolated cells, the alpha 2 Na pump subunit is not detected in AT1 cells, although a strong band is seen with the PCR-amplified alpha 2-subunit plasmid. No bands are seen following reverse transcription without PCR.


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Fig. 8.   Expression of Na pump and Na channel subunits in isolated AT1 cells by RT-PCR. RNA from highly purified (~95%) AT1 cells was reverse transcribed and subjected to PCR using primers specific for the Na pump and Na channel subunits. The alpha 1- and beta 1-subunits of Na-K-ATPase and alpha -rENaC are detected in AT1 cells (lanes 1, 6, and 11). Faint bands are seen in lanes derived from RNA from AT2 cells equal to the number of contaminating cells present in AT1 cell preparations (lanes 3, 8, and 13). The alpha 2-subunit of Na-K-ATPase is not detected in either AT1 cells (lane 16) or AT2 cells (lane 18). Lanes 2, 4, 7, 9, 12, 14, 17, and 19 are negative controls in which no bands are seen without reverse transcription for the Na pump alpha 1- and beta 1-subunits, alpha -rENaC, and Na pump alpha 2-subunit. Lanes 5, 10, 15, and 20 are positive controls using PCR-amplified plasmids for Na pump alpha 1- and beta 1-subunits, alpha -rENaC, and Na pump alpha 2-subunit. Lane M represents a 1,000-bp molecular weight marker.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We evaluated expression of Na pump and Na channel subunits in isolated AT1 cells and in AT1 cells in situ. Utilizing a panel of cell-specific antibodies to identify AT1 cells within partially purified populations of freshly isolated distal lung cells, we demonstrated that isolated cells labeled by VIIIB2 are also reactive with anti-AQP-5. Furthermore, cells identified by these AT1 cell-specific antibodies do not react with anti-SP-A or anti-LCA, demonstrating the success of this approach for identification of AT1 cells within mixed alveolar cell populations. Using a strategy of concurrent labeling and immunofluorescent detection, we localized Na channel and Na pump subunits to isolated cells that are reactive with the AT1 cell-specific antibodies. Expression of the alpha 1- and beta 1-subunits, but not the alpha 2-subunit isoforms of Na-K-ATPase, and the alpha -subunit of rENaC was demonstrated in isolated AT1 cells. Consistent with these IF results, transcripts for the Na pump alpha 1- and beta 1- (but not alpha 2-) subunits and alpha -rENaC were also detected in highly purified AT1 cells by RT-PCR. Concurrent labeling with anti-AQP-5 in whole lung demonstrates expression of the alpha 1 Na pump subunit by AT1 cells in situ. These findings represent the first successful demonstration that AT1 cells express Na transport proteins, suggesting a role for AT1 cells in Na transport across alveolar epithelium.

Until recently, AT1 cells have largely been characterized by their morphological appearance. Because AT1 cell morphology changes markedly following cell isolation, definitive identification of isolated AT1 cells has been problematic. The advent of a number of antibody probes that react specifically with AT1 cells in the adult rat lung in situ has provided nonmorphological means to identify AT1 cells in vivo and, as methods for isolation of AT1 cells, have been developed, within populations of isolated lung cells. VIIIB2 is a murine MAb generated in our laboratory that recognizes an epitope in the apical membrane of AT1 cells that is not detected in other rat lung or nonlung cells (13). The polyclonal anti-AQP-5 antibody recognizes a 28-kDa water channel present in the adult in salivary and lacrimal tissues and in lung (7, 25, 38). In the alveolar epithelium, AQP-5 is expressed only on the apical surface of AT1 cells (30). Using these antibodies for labeling of cytocentrifuged preparations of isolated alveolar cells, colocalization of these antibodies that are specific for AT1 cells in situ is seen. Furthermore, cells that are reactive with anti-SP-A (AT2 cells) or anti-LCA (leukocytes) are not reactive with the AT1 cell-specific antibodies, consistent with their identity as AT1 cells.

Several methods have been described for isolation of AT1 cells. Approaches have included digestion using different combinations of enzymes in conjunction with density gradient separation (53), centrifugal elutriation (1), and/or selective adhesion. However, results have been variable, and routine isolation of large numbers of viable AT1 cells to high levels of purity has been problematic. In part, this may reflect the inability to easily detach these large cells, with their expansive cytoplasmic processes, from the underlying basement membrane. Most recently, using elastase at 4.5 U/ml, together with subsequent magnetic selection with cell-specific antibodies, isolation of AT1 cells with purity ranging from 60-86% has been reported (15). Perhaps as a consequence of the large surface over which they are spread, and to release AT1 cells from the basement membrane, elastase was used at more than twice the concentration usually required for AT2 cell isolation. In addition, to allow easy passage of the larger AT1 cells, filtration steps were performed using Nitex mesh of relatively large pore size (20 µm). Using a modification of this approach, we successfully isolated mixed cell populations comprised of approximately equal numbers of AT1 cells, AT2 cells, and leukocytes. We demonstrated that using a strategy in which cells are concurrently labeled with AT1 cell-specific antibodies, proteins of interest can successfully be localized to individual AT1 cells. Nevertheless, since this level of purity is not adequate for analysis of mRNA in AT1 cells, immunomagnetic selection was undertaken using the AT1 cell-specific MAb VIIIB2 in conjunction with a secondary antibody conjugated to magnetic beads. This approach yielded AT1 cells of sufficient purity (>= 95%) for analysis of mRNA expression by RT-PCR. However, immunoselection with MAb VIIIB2 precluded the concurrent use of Na pump subunit MAbs for colocalization in highly purified AT1 cell populations, making it preferable to perform the double labeling IF experiments with partially purified cell preparations.

Na-K-ATPase is a heterodimeric protein consisting of an alpha - and a beta -subunit. The Na pump subunits occur in one of several distinct isoforms having characteristic tissue distribution (34). There is general agreement that the predominant isoforms expressed in lung are alpha 1- and beta 1-isoforms, which have been localized to both AT2 cells in situ (19, 45) and isolated AT2 cells (29, 33, 41, 48). Although low levels of the alpha 2-subunit isoform have been detected in whole lung (34, 54), the precise distribution, if any, of this isoform within cells of the alveolar epithelium has not been resolved (3, 40). Although it is generally accepted that AT2 cells possess functional Na pumps, for reasons delineated further below, previous attempts to localize antigenic Na pumps to AT1 cells in situ have been unsuccessful to date (19, 45). The IF and RT-PCR data in this study in freshly isolated cells indicate that the alpha 1- and beta 1-subunits, but not the alpha 2-subunit, of Na-K-ATPase are in fact expressed by AT1 cells, while expression of the alpha 1-subunit was also confirmed in AT1 cells in situ. Although we demonstrate here expression of the alpha 1 and beta 1 Na pump subunits in AT1 cells, further studies are needed to delineate their functional role in these cells.

mRNA for all three subunits of the rENaC have been identified in adult rat lung (27, 39, 52), in freshly isolated rat AT2 cells (32, 49), and in AEC after several days in culture (3, 12). In situ hybridization demonstrates diffuse labeling of the distal alveolar surface for predominantly the alpha - and gamma -subunits of rENaC (16, 27, 52), although none of these subunits has been detected to date by immunohistochemistry in distal rat lung (39). The pattern of labeling of the alveolar surface for alpha -rENaC by in situ hybridization has been interpreted to be consistent with labeling primarily of AT2 cells, but the diffuse nature of the signal makes it impossible to conclude that AT1 cells do not express alpha -rENaC. We demonstrate in the current study that the alpha -subunit of rENaC is expressed by freshly isolated AT1 cells. Consistent with our findings in isolated cells, a recent preliminary study by Johnson et al. (21) reported that all three Na channel subunits are present by IF in AT1 cells in whole lung. Although we did not evaluate rENaC function in this study, further work will be needed to determine its functional properties in these cells.

The large surface area occupied by AT1 cells in the lung, in conjunction with observations that alveolar fluid clearance is both ouabain sensitive and amiloride inhibitable, has always made it seem unlikely that AT1 cells do not express Na pumps and Na channels. Failure to detect antigenic Na pumps in AT1 cells in situ in previous studies is thought to perhaps reflect the fact that Na pumps in AT1 cells are present at lower antigenic density than in AT2 cells (due perhaps to their larger surface area) at a level below the sensitivity of detection with the methods previously employed (45). Lower antigenic density in AT1 cells than in AT2 cells is supported by our observations that although the alpha 1-subunit is detected in AT1 cells in situ in the current study, relative signal intensity is far stronger in AT2 cells than in adjacent AT1 cells in whole lung (Fig. 7C). Increased sensitivity for detection of Na pump subunits in isolated AT1 cells compared with whole lung may reflect, in part, differences in the methods of fixation used for isolated cells compared with whole lung. Reduction of antibody binding by fixation (especially with aldehyde fixatives) was suggested by Schneeberger and McCarthy (45) as a possible explanation for the patchy detection of Na pumps in AT2 cells in whole lung and for failure to detect antigenic Na pumps at all in AT1 cells. In addition, following isolation, the cells round up, probably resulting in a higher antigenic density per apparent membrane surface area than is present in situ. Induction of Na transporter proteins during the isolation procedure, while possible, seems an unlikely explanation given that the cells in the partially purified preparations undergo very few manipulations in the short time before fixation. In any event, given that Na pump subunits in AT1 cells may be present at a lower antigenic density than in AT2 cells, availability of different antibodies in conjunction with the use of antigen retrieval and signal amplification approaches likely resulted in increased sensitivity, allowing us to identify the antigenic signal in AT1 cells in situ.

Besides the recent demonstration that AT1 cells are highly permeable to water, suggesting a role for AT1 cells in water transport across alveolar epithelium (15), and a presumptive role in gas exchange, knowledge of AT1 cell function and identification of the genes and proteins expressed by these cells has been limited (44). Characterization of AT1 cell function and patterns of gene expression have been hindered by the difficulty in isolating purified populations of AT1 cells and in identifying isolated AT1 cells as well as AT1 cells in situ definitively by nonmorphological means. In this study, using complementary approaches in isolated cells and whole lung, we successfully localized both Na pump and Na channel subunits for the first time to AT1 cells, implicating AT1 cells in active Na transport across the alveolar epithelium.


    ACKNOWLEDGEMENTS

This work was supported in part by the American Lung Association, the American Heart Association, National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, HL-38658, HL-51928, HL-64365, and HL-62569, the Baxter Foundation, and the Hastings Foundation.


    FOOTNOTES

E. D. Crandall is Hastings Professor and Norris Chair of Medicine.

Address for reprint requests and other correspondence: Z. Borok, Division of Pulmonary and Critical Care Medicine, Univ. of Southern California, IRD 602, 2020 Zonal Ave., Los Angeles, CA 90033 (E-mail: zborok{at}usc.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.

10.1152/ajplung.00130.2000

Received 21 April 2000; accepted in final form 27 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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