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Published ahead of print on May 14, 2003, doi:10.1165/rcmb.2002-0281OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 552-561, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0281OC

Cell–Cell Communication in Heterocellular Cultures of Alveolar Epithelial Cells

Brant E. Isakson, Gregory J. Seedorf, Richard L. Lubman, W. Howard Evans and Scott Boitano

Department of Physiology, Arizona Respiratory Center, University of Arizona Health Sciences Center, Tucson, Arizona; Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming; Division of Pulmonary and Critical Care Medicine, University of Southern California Keck School of Medicine, Los Angeles, California; and Department of Medical Biochemistry, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, Wales, United Kingdom

Address correspondence to: Scott Boitano, Arizona Respiratory Center, Department of Physiology, University of Arizona Health Sciences Center, Room 2341 AHSC, 1501 N. Campbell Ave., Tucson, AZ 85724. E-mail: sboitano{at}email.arizona.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mammalian alveolar epithelium is composed of alveolar type I (AT1) and alveolar type II (AT2) cells that together coordinate tissue function. We used a heterocellular culture model of AT1 and AT2 cells to determine pathways for intercellular signaling between these two phenotypes. Gap junction protein (connexin) profiles of AT1 and AT2 cells in heterocellular cultures were similar to those seen in rat lung alveolar sections. Dye coupling studies revealed functional gap junctions between and among each cell phenotype. Localized mechanical stimulation resulted in propagated changes of intracellular Ca2+ to AT1 or AT2 cells independent of the stimulated cell phenotype. Ca2+ communication that originated after AT1 cell stimulation was inhibited by gap junction blockers, but not by an inhibitor of extracellular nucleotide signaling (apyrase). Conversely, Ca2+ communication after stimulation of AT2 cells was not significantly reduced by gap junction inhibitors. However, apyrase significantly reduced Ca2+ communication from AT2 to AT1 cells, but not from AT2 to AT2 cells. In conclusion, AT1 and AT2 cells have unique connexin profiles that allow for functional coupling and distinct intercellular pathways for coordination of Ca2+ signaling.

Abbreviations: Alexa Fluor 350, A350 • 18 {alpha}-glycyrrhetinic acid, {alpha}-GA • alveolar type I pneumocyte, AT1 cell • alveolar type II pneumocyte, AT2 cell • intracellular Ca2+ concentration, [Ca2+]i • connexin, cx • Dulbecco's Modified Eagle's Medium, DMEM • 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid, 5Na, Fura-2 • Hanks' Balanced Saline Solution, HBSS • Lucifer Yellow, LY • phosphate-buffered saline, PBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The alveolar epithelium provides the physical barrier that separates the gaseous alveolar space from the aqueous internal milieu. It consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells, present in approximately equal numbers. AT1 cells display a flattened morphology and comprise greater than 90% of the alveolar surface area, whereas cuboidal AT2 cells are generally located in the corners of the alveoli (1). AT1 cells facilitate gas and water exchange across the epithelial barrier (2). AT2 cells secrete surfactant, contribute to ion and fluid transport, are progenitors for both AT1 and AT2 cells, and may provide innate immunity within the lung (3–5).

Due to the complex architecture of the lung and the difficulty in harvesting AT1 cells, isolated AT2 cells are the most common in vitro model for study of alveolar epithelial cell function. However, within days of tissue culture, primary cultures of AT2 cells adopt an AT1-like phenotype (6) unless specialized techniques for maintaining AT2 phenotype are used (e.g., Refs. 7–13). These culture methods have led to increased knowledge of the functional aspects of AT2 cells. In vivo, however, AT1 and AT2 cells are intermixed and cooperate with each other to ensure integrated physiologic function for lung epithelium. In this study, an alveolar epithelial culture that displays functional junctions linking AT1-like and AT2-like cells after 7 d in culture was used to model communication in the alveolar epithelium (14).

Tight junctions and gap junctions were first identified between pulmonary alveolar epithelial cells by electron microscopy (15, 16). AT1 and AT2 cells from mammalian lung alveoli in situ have been shown to express gap junction proteins (connexins) (17, 18). Many studies on connexin expression in alveolar tissue and alveolar epithelial cell cultures have uncovered complex patterns of protein expression with at least six different connexins (Cxs): Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 (7, 11, 19–24). Changes in connexin expression in alveolar epithelial cells have been associated with developing or post-injury airways (19, 21, 25), phenotypic changes inherent in alveolar cell culture systems (7, 11, 24), or changes in extracellular signaling molecules, including growth factors and matrix proteins (7, 11, 22, 26). Although alveolar epithelial cells display a complex regulation of connexin expression, it is unclear how or whether gap junctional communication contributes to physiologic changes in the lung.

Stretch-induced Ca2+ transients in alveolar epithelial cells (27) suggested that changes in intracellular Ca2+ concentration ([Ca2+]i) can contribute to integrated signaling in lung epithelium. Indeed, Ca2+ signaling is associated with surfactant secretion (28, 29), extracellular matrix protein synthesis (30), recognition of AT1 cell signaling (18), selective alveolar epithelial cell death (31), and cell differentiation (32). Cultured airway epithelial cells can communicate [Ca2+]i changes between neighboring cells (11, 23, 24, 33, 34), presumably to coordinate cellular function into overall tissue response. At least two mechanisms underlie the coordination of [Ca2+]i changes among neighboring lung epithelial cells: diffusion of second messenger molecules/ions through gap junctions and release of ATP or UTP into extracellular spaces with subsequent activation of Ca2+ signaling pathways via plasma membrane purinergic receptors. In homogenous primary cultures of alveolar epithelial cells (i.e., one predominate phenotype), AT1-like and AT2-like cells have independently been shown to increase [Ca2+]i in response to ATP or UTP, and are functionally coupled via gap junctions that allow transfer of small fluorescent dyes (7, 11, 19, 23–35). Also, both cell types have been shown to coordinate [Ca2+]i changes among neighboring cells in culture. However, the biochemical mechanisms facilitating this Ca2+ communication in homogenous cultures of AT1-like or AT2-like cells are different. AT1-like cells coordinate changes in [Ca2+]i via gap junctional communication (23), and AT2 cells coordinate changes via extracellular nucleotide release (11). The specificity of second messenger signaling may correspond with the distinctive connexin profiles expressed by AT1-like and AT2-like cells (11, 23).

In this study, connexin expression patterns in alveolar lung sections and heterocellular cultures of alveolar epithelial cells were studied by immunocytochemistry. Additionally, dye coupling and second messenger (Ca2+) communication between and among each cell phenotype were explored using this heterocellular culture model. Connexin expression in AT1-like and AT2-like cells in heterocellular cultures were similar to that seen in alveolar sections, with each cell phenotype displaying distinct patterns of dye transfer and Ca2+ communication that may be related to changes in the connexin profile. The different intercellular signaling properties of AT1-like and AT2-like cells in heterocellular cultures may provide insight to how AT1 and AT2 cells interact to regulate tissue function in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Growth medium, fetal bovine serum, and trypsin inhibitor were from Gibco BRL (Rockville, MD). Alexa Fluor 350 (A350) (MW = 349; one negative charge) was from Molecular Probes (Eugene, OR). 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid, 5Na (Fura 2)-acetoxymethyl ester (Fura 2-AM) and Fura 2 were from CalBiochem (La Jolla, CA). ATP (A 2,383), UTP (U 6,875), fibronectin, DNase, IgG, apyrase, 18 {alpha}-glycyrrhetinic acid, and Lucifer Yellow (LY) (MW = 457; two negative charges) were from Sigma Chemical (St. Louis, MO). Collagen was isolated from rat tails as described (14). Laminin-5 was collected from 804G cell culture supernatants (36). The 804G cell line was kindly provided by Dr. J. C. R. Jones (Northwestern University). Elastase was from Worthington Biochemical (Lakewood, NJ). Gap junctional mimetic peptide inhibitors, gap26 (amino acid sequence: VCYDKSFPISHVR) and gap27 (amino acid sequence: SRPTEKTIFII) were synthesized by Alpha Diagnostics International (Houston, TX). All other chemicals were purchased through Fisher (Houston, TX) or VWR (Denver, CO) and were of the highest biochemical grade.

Antibodies
Rabbit antipeptide antibodies to Cx37, Cx40, Cx46 were from ADI, Inc. (San Antonio, TX). Antibodies to Cx26, Cx32, and Cx43 were developed in our laboratory (W.H.E.); these antibodies have been fully characterized (37, 38). A second antibody to Cx43 and all FITC- and Cy5-labeled goat anti-rabbit IgG secondary antibodies were from Chemicon Corporation (Temecula, CA).

Lung Alveolar Section Preparation and Staining
Male Sprague-Dawley rats (250–300 g) were injected with sodium pentobarbital, and their lungs surgically exposed and washed with solution I (136 mM NaCl, 2.2 mM Na2HPO4, 5.3 mM KCl, 5.6 mM glucose, and 10 mM HEPES, pH 7.4) to remove blood. Lungs were placed in a 10% formalin solution in phosphate-buffered saline (PBS: 8 mM Na2HPO4, 128 mM NaCl, 2 mM KCl, 10 mM HEPES, pH 7.4) for at least 20 min, and 1-mm-thick sections were prepared. After 12 h in 10% formalin solution, sections were dehydrated by sequential washes: 1 h in 80% EtOH; 2x 1 h in 95% EtOH; 2x 1 h in 100% EtOH; 1 h in xylene; and 2 h in xylene. Sections were immersed with three changes of 58°C paraffin over 3 h and embedded. After washing, 4-µm sections (Ergostar Micron HM200 microtome, San Marcos, CA) were mounted onto glass slides and dried at 37°C overnight. Slides were deparaffinized by sequential washes: 3x 5 min xylene; 2x 1 min 100% EtOH; 2x 1 min 95% EtOH; 1x 70% EtOH, and were immersed in sterile deionized H2O for at least 15 min before immunohistochemistry. Sections were subjected to heat-induced epitope retrieval by boiling in citrate buffer (1.8 mM C6H8O7, 8.2 mM C6H5Na3O7) for 10 min. Sections were then washed at room temperature with 0.5% Tween-20 in PBS (PBST) for 5 min, twice for 5 min in PBS, and once for 2 h in 3% BSA in PBST (PBSAT). Sections were then incubated with primary anti-Cx antibody overnight at 4°C and washed (2x 30 min PBSAT; 4x 5 min PBS; 1x 2 h PBSAT) before incubation with secondary antibody for 1 h at 37°C. Sections were washed further (2x 30 min PBSAT; 4x 5 min PBS; and 2x 2 min deionized H2O) and mounted for confocal microscopy. Differential interference contrast and confocal images were obtained on a inverted Leica TCS-SP2 confocal laser scanning microscope with a x100, 1.4 N.A. oil immersion objective.

Heterocellular Alveolar Cell Cultures
Cells were cultured on rat tail collagen/fibronectin/laminin-5 coated glass coverslips (14). Briefly, rat tail collagen (0.5 ml) was mixed with 50 µl of flavin mononucleotide and a 50-µl aliquot of this mixture immediately spread evenly across a 15-mm glass coverslip and solidified by illumination with a full spectrum lamp. Coverslips were sequentially washed 3x 10 min in deionized H2O and 1x 20 min in Hanks' Balanced Saline Solution (HBSS; 1.3 mM CaCl2, 5.0 mM KCl, 0.3 mM KH2PO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 137.9 mM NaCl, 0.3 mM Na2PO4, and 1% glucose buffered with 25 mM HEPES, pH 7.4) to remove flavin mononucleotide. Before cell plating, coverslips were incubated in 300 µl of 50 µg/ml fibronectin at 4°C for 1 h. Coverslips were warmed to room temperature and treated with 1 µl of laminin-5 rich supernatant obtained from the rat 804G cell line deposited at the center of the coverslip and left at 37°C for 1 h. Coverslips were then immersed in serum-free Dulbecco's Modified Eagle's Medium/F-12 (DMEM/F-12) until cell plating (within 1 h of preparation).

Primary AT2 cells were isolated from lungs of male Sprague-Dawley rats (250–300 g). The lungs were perfused with solution I, removed from the animal, and lavaged with solution II (136 mM NaCl, 2.2 mM Na2HPO4, 5.3 mM KCl, 5.6 mM glucose, 1.9 mM CaCl2, 1.3 mM MgSO4, and 10 mM HEPES, pH 7.4) supplemented with ~ 4 U/ml elastase. Each rat lung was minced in a trypsin inhibitor solution (10 ml solution I supplemented with 100 mg BSA, 10 mg trypsin inhibitor, 10 mg DNase, and a final concentration of 0.4 mM EDTA) and panned on IgG-coated plates for 1 h at 37°C. Cells were recovered and plated on matrix-coated coverslips at a density of ~ 1.8 x 106 cells/ml. Cell viability was assayed by 5% trypan blue exclusion and exceeded 90%. Cells were grown in DMEM/F-12 supplemented with 10% fetal bovine serum, penicillin, streptomycin, and amphotericin B at 37°C in a 5% CO2 atmosphere for 7 d. Culture medium was changed at Day 1, and every other day thereafter.

Immunocytochemistry of Heterocellular Cultures
Cell cultures were washed twice for 5 min with PBS and fixed for 10 min with 3.7% formaldehyde in PBS. Coverslips were washed with PBS, incubated with PBST for 5 min, and washed with PBSAT for 2 h at room temperature. Cells were incubated with the appropriate primary antibody for 2 h in PBSAT at 37°C, washed (2x 30 min with PBSAT; 4x 5 min with PBS), and incubated with secondary antibody for 1 h at 37°C. The preparations were again washed (2x 30 min with PBSAT; 4x 5 min with PBS; 2x 5 min deionized H2O) and mounted for observation.

Dye Coupling
Seven-day-old heterocellular cultures were washed thoroughly at room temperature in HBSS and placed in 100-mm petri dishes containing HBSS. Eppendorf (Brinkmann, Westbury, NY) femptotips were backfilled with 10 mM LY or A350 in 200 mM KCl. AT1-like or AT2-like cells were chosen by morphologic characteristics. Microinjection (Eppendorf Micromanipulator 5,171 and Transjector 5,426) into the cytoplasm of individual cells and subsequent dye transfer were monitored on a Nikon Eclipse TE300 (Melville, NY) or an Olympus IX70 (Melville, NY) inverted microscope with x20 objective in phase contrast for injections and in epifluorescence mode for dye coupling. Images were captured immediately after injection and at 5 min after injection with a CoolSnap camera using Roper Scientific imaging software (Tucson, AZ). Cells were considered to be functionally coupled if two or more neighboring cells displayed fluorescence within 5 min of dye injection. Inhibitors (in HBSS) were applied for 45 min at the following concentrations: {alpha}-glycyrrhetinic acid, 20 µM; gap27 130 µM; gap26 160 µM. Addition of apyrase had no effect on dye transfer experiments.

Ca2+ Communication
Seven-day-old heterocellular cultures were incubated in 5 µM Fura 2-AM in HBSS for 75 min, washed for 20 min in HBSS, and observed on an inverted Olympus IX70 microscope. Excitation of Fura 2 was alternated between 340 and 380 nm using a Delta Ram illuminator (Photon Technologies, Lawrenceville, NJ). Images of emitted fluorescence > 505 nm were recorded by an ICCD camera (Photon Technologies) under software control (Image Master; Photon Technologies). Calculations of [Ca2+]i were by published equations (39). Mechanical stimulation was by glass micropipette (tip diameter ~ 1 µm) under piezoelectric control, positioned using a micromanipulator (SD Instruments, Grants Pass, OR), and deflected downward for 150 ms to deform an individual cell. To preclude wound-induced cellular signaling (23) only experiments where the stimulated cell membrane was left intact (as determined by phase-contrast observation and Fura 2 dye retention measured at 340 nm excitation) were included in the analysis. Changes in [Ca2+]i of at least 150 nM (2- to 3-fold change over resting values) were considered positive [Ca2+]i changes. Cells were considered to communicate Ca2+ changes if two or more neighboring cells displayed positive [Ca2+]i changes within 20 s. Addition of apyrase did not alter resting [Ca2+]i of the heteroculture cells. Addition of gap junction inhibitors ({alpha}-glycyrrhetinic acid, gap26 or gap27) did not alter resting [Ca2+]i nor the response to exogenous ATP of AT1-like or AT2-like cells in culture. Addition of the intracellular loop mimetic peptide, des5 (40), had no effect on resting [Ca2+]i or coordination of [Ca2+]i changes between or among AT1-like and AT2-like cells.

Statistics
Functional dye coupling and Ca2+ communication between individual cells were tested for equality and significant differences between variables were determined using binary population proportion statistics. Histograms display percent of experiments with positive dye coupling or Ca2+ communication ± SE. In comparisons between experimental paradigms, a statistical value of P < 0.05 was used to establish significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Connexin Expression in Lung Alveolar Slices
We used immunocytochemistry and laser scanning confocal microscopy of rat alveolar lung sections stained with connexin-specific antibodies to determine connexin isoform expression in situ (Figure 1). Sections treated with anti-Cx26 (Figures 1A–1B) displayed relatively light staining throughout the alveolus. Sections treated with anti-Cx32 (Figures 1C–1D) or anti-Cx40 (Figures 1G–1H) also displayed a light staining pattern; however, there were frequently rounded patterns of staining that appeared to be associated with areas of AT2 cells with these antibodies. Staining for Cx37 was negative in all alveolar epithelial samples (Figures 1E–1F). Sections treated with anti-Cx43 (Figures 1I–1J) were the most strongly immunoreactive, displaying staining throughout the alveolus. Sections stained with anti-Cx46 (Figures 1K–1L) were also highly immunoreactive throughout the alveolus.



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  		Figure 1. Connexin expression in lung alveolar sections. Differential interference contrast and immunocytochemical staining of individual connexin (Cx) isoforms are shown for six connexins. Images are of sections immunostained with antibodies specific for: (A and B) Cx26; (C and D) Cx32; (E and F) Cx37; (G and H) Cx40; (I and J) Cx43; and (K and L) Cx46. Bar in A indicates 10 µm, and is representative of all images. In alveolar sections Cx26, Cx32, and Cx40 display a diffuse staining pattern with areas of heavy immunostaining. Cx43 and Cx46 stain throughout the alveolus. No staining for Cx37 was evident in the alveolar epithelium.

 
Connexin Expression in Heterocellular Alveolar Cultures
To compare in vitro connexin profiles with the in situ patterns of the alveolar epithelial sections, heterocellular cultures with morphologically distinct AT1-like and AT2-like cells (14) were evaluated for individual connexin expression. Immunocytochemical detection of both Cx26 (Figures 2A–2B) and Cx32 (Figures 2C–2D) resulted in a diffuse staining pattern in areas with both AT1-like and AT2-like cells. There was no staining of Cx37 in either cell type (Figures 2E–2F). Cultures stained for Cx40 displayed a distinct pattern: areas with AT1-like cells were devoid of staining and areas with AT2-like cells were highly immunoreactive (Figures 2G–2H). Two separate antibodies for Cx43 displayed staining in areas with AT1-like or AT2 like cells (Figures 2I–2J). Antibodies to Cx46 were most immunoreactive in areas with AT1-like cells (Figures 2K–2L), however, up to 20% of the AT2-like cells in heterocellular culture also displayed staining for Cx46 (data not shown). The overall connexin profile observed for AT1-like and AT2-like cells in heterocellular cultures was similar to that seen in alveolar slices (Figures 1 and 2).



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  		Figure 2. Connexin expression in heterocellular cultured alveolar epithelial cells. Seven-day-old heterocellular cultures of alveolar epithelial cells were stained with antibodies specific for: (A and B) Cx26; (C and D) Cx32; (E and F) Cx37; (G and H) Cx40; (I and J) Cx43; and (K and L) Cx46. Bar in A indicates 10 µm. AT1-like and AT2-like cells are identifiable by morphologic characteristics in each image pair; arrows indicate AT2-like cells. Areas with AT1-like cells display staining for Cx26, Cx32, Cx43, and Cx46. Areas with AT2-like cells display staining for Cx32, Cx40, and Cx43. Neither cell type stained for Cx37.

 
Dye Coupling in Alveolar Epithelial Heterocellular Cultures
The immunocytochemical detection of connexins in alveolar epithelial sections and heterocellular cultures provided the incentive to investigate whether cells were using gap junctions for intercellular communication. AT1-like or AT2-like cells in heterocellular cultures were microinjected with the anionic dyes LY or A350 and diffusion was monitored 5 min after injection (Table 1). Microinjection of AT1-like cells with LY indicated a high incidence of dye coupling to both neighboring AT1-like or AT2-like cells (Figures 3A–3C). Dye coupling after microinjection of AT2-like cells with LY also resulted in a high incidence of dye coupling to neighboring AT2-like cells, however, diffusion of LY from AT2-like cells to neighboring AT1-like cells was significantly reduced (Figures 3A, 3D, and 3E).


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  		TABLE 1 Summary data for dye and second messenger coupling of alveolar epithelial cells

 


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  		Figure 3. LY dye transfer between alveolar epithelial cells in heterocellular cultures. In each experiment, LY was microinjected into a single cell and neighboring cells were assayed at 5 min for dye transfer. Coupling occurred when two or more neighboring cells took up LY in this time period. (A) Summary data for microinjection of AT1-like cells and transfer to AT1-like cells (dark gray columns); AT1-like to AT2-like (light gray columns); AT2-like to AT2-like (open columns); and AT2-like to AT1-like (black columns) under control (no treatment) and inhibitor conditions ({alpha}-GA = 18 {alpha}-glycyrrhetinic acid, gap27 and gap26 are connexin mimetic peptides). Columns (± SE) represent the percentage of experiments with dye transfer to at least two neighboring cells of the represented phenotype. "#" indicates significant reduction in LY transfer from AT2-like to AT1-like cells compared with other three injection paradigms in control conditions; "*" indicates significant difference in LY transfer between control and given inhibitor (both values are P < 0.05). Micrographs are representative phase contrast/LY fluorescence image pairs of each of the experimental conditions described above. Microinjected cells are marked with an "*" and AT2-like cells that receive LY are marked with a "2" in the confocal micrographs. Bar in C indicates 10 µm, and is indicative for all images. LY injections of AT1-like cells resulted in a high incidence of functional dye coupling to AT1-like cells or to AT2-like cells (A–C) that was inhibited by all three inhibitors (A, F, G, J, K, N, and O). LY injections of AT2-like cells also resulted in a high incidence of dye transfer to AT2-like cells; however, functional coupling of LY from AT2-like cells to AT1-like cells was significantly reduced from the three other experimental paradigms (A, D, and E). The connexin mimetic peptide inhibitors, but not 18 {alpha}-GA, significantly reduced dye transfer from AT2-like cells (A, H, I, L, M, P, and Q). See text and Table 1 for further details.

 
To inhibit dye transfer, heterocellular cultures were initially exposed to 18 {alpha}-glycyrrhetinic acid. Although LY dye transfer was reduced when compared with inhibitor-free conditions, a low level of transfer persisted (Figures 3A, 3F–3I) and only transfer from AT1-like to AT1-like was significantly lower than control. To further evaluate the role of gap junctions in dye transfer, the connexin mimetic peptide inhibitor gap27 was applied to heterocellular cultures (41). This peptide corresponds to a specific region of the second extracellular loop in rat Cx43 that displays some homology with all of the connexins studied (Table 2). Incubation in the presence of gap27 resulted in a significant reduction in dye transfer between AT1-like cells, but LY dye transfer from AT1-like cells to AT2-like cells was more persistent (Figures 3A, 3J, and 3K). LY dye transfer after microinjection of AT2-like cells was effectively inhibited by gap27 (Figures 3A, 3L, and 3M). Because connexin mimetic peptides may not be equally potent inhibitors of gap junctions made up of different connexin isoforms owing to minor variations in the extracellular loop sequences (42), a second connexin mimetic protein derived from the first extracellular loop region of rat Cx43 (gap26; Table 2) was used (Figures 3A, 3N–3Q). In these experiments, microinjection of AT1-like cells resulted in significantly reduced LY coupling to AT1-like or AT2-like cells. Transfer of LY after microinjection of AT2-like cells to either AT1-like cells or AT2-like cells was also significantly reduced when compared with control.


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  		TABLE 2 Amino acid sequences for connexin mimetic peptide inhibitors and corresponding regions in connexin isoforms expressed in alveolar epithelial cells

 
To further evaluate dye coupling in heterocellular cultures, individual cells were microinjected with A350, a dye of lower molecular weight and decreased negative charge than LY. Microinjection of AT1-like cells resulted in a similar high incidence of dye coupling to AT1-like cells and to AT2-like cells (Figures 4A–4C). AT2-like cells also transferred A350 to AT1-like and AT2-like cells (Figures 4A, 4D, and 4E); both of these coupling indices were significantly higher than that observed after microinjection of AT2-like cells with LY (Table 1; Figures 3 and 4). To further analyze A350 dye coupling, microinjections were performed in the presence of various gap junction inhibitors. Incubation of the heterocellular cultures with 18 {alpha}-glycyrrhetinic acid significantly reduced A350 dye coupling, however, coupling was still evident in 33% of each experimental condition (Figures 4A, 4F–4I). The effects of gap27 and gap26 on A350 dye transfer were similar to that seen with LY experiments, with gap27 significantly lowering dye transfer between AT1-like cells but not from AT1-like to AT2-like cells (Figures 4A, 4J, and 4K). Microinjection of AT2-like cells in the presence of gap 27 resulted in significantly reduced transfer to both AT1-like and AT2-like cells (Figures 4A, 4L, and 4M). Microinjection of AT1-like cells in the presence of gap26 significantly lowered dye transfer to both AT1-like cells and to AT2-like cells (Figures 4A, 4N, and 4O). Microinjection of AT2-like cells in the presence of gap 26 resulted in significantly reduced transfer to both AT1-like and AT2-like cells (Figures 4A, 4P, and 4Q). Although the three gap junction inhibitors studied reduced dye transfer, gap26 was the most effective inhibitor of dye coupling.



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  		Figure 4. A350 dye transfer between cultured alveolar epithelial cells in heterocellular cultures. Experiments and column notations are as in Figure 3; additionally, "^" indicates significant difference compared with LY coupling experiments with similar paradigms (P < 0.05). (A) Summary data for microinjection of AT1-like cells and transfer to AT1-like cells (dark gray columns); AT1-like to AT2-like (light gray columns); AT2-like to AT2-like (open columns); and AT2-like to AT1-like (black columns) under control (no treatment) and inhibitor conditions. A350 injection into AT1-like cells resulted in a high incidence of functional dye coupling to AT1-like cells and AT2-like cells (A–C) as with LY injection (Figure 3). A350 injection into AT2-like cells resulted in significantly higher incidence of cell coupling to AT1-like cells and AT2-like cells (A, D, and E) than that seen after LY injection. All three inhibitors significantly reduced dye transfer from AT1-like to AT2-like cells and from AT2-like cells to either phenotype (A, F–Q). There was a significant reduction in A350 transfer from AT1-like cells to AT2-like cells in the presence of 18 {alpha}-GA and gap26; however, the reduction by gap27 (A, J, and K) was not significantly different than the control conditions. See text and Table 1 for further details.

 
Ca2+ Communication in Heterocellular Alveolar Cell Cultures
The dye coupling experiments described above showed coupling between all cells in the heterocellular cultures but suggested that differential gap junctional coupling occurred between cell phenotypes. These studies were extended to determine if alveolar cells could communicate physiologic changes to neighboring cells in heterocellular cultures. Alveolar epithelial cells in homogenous cultures respond to localized mechanical stimulation by increasing [Ca2+]i that is propagated to adjacent cells by either gap junctional (AT1-like cells) or paracrine (AT2-like cells) mechanisms (11, 23, 24). To assess whether heterocellular cultures of alveolar epithelial cells propagated such intercellular Ca2+ waves, a single alveolar epithelial cell was mechanically stimulated and increases of [Ca2+]i in neighboring cells evaluated (Figure 5; Table 1). Mechanical stimulation of an AT1-like cell resulted in an elevated [Ca2+]i that was propagated to both AT1-like and AT2-like cells. Similarly, mechanical stimulation of an AT2-like cell resulted in an high incidence of propagated increases of [Ca2+]i to both AT1-like and AT2-like cells.



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  		Figure 5. Ca2+ communication in heterocellular cultures of alveolar epithelial cells. Mechanical stimulation of a single cell was used to initiate an increase in [Ca2+]i in the stimulated cell and subsequent changes in [Ca2+]i in neighboring cells. Columns represent the percentage of experiments (± SE) where mechanical stimulation resulted in an increase in [Ca2+]i in two or more neighboring cells for each paradigm initially described in Figure 3. (A) Summary data for microinjection of AT1-like cells and transfer to AT1-like cells (dark gray columns); AT1-like to AT2-like (light gray columns); AT2-like to AT2-like (open columns); and AT2-like to AT1-like (black columns) under control (no treatment) and inhibitor conditions. "*" indicates significant difference from similar experimental paradigm in absence of inhibitor (P < 0.05). Mechanical stimulation of an AT1-like cell resulted in a high incidence of propagated [Ca2+]i changes to AT1-like cells or AT2-like cells. Similarly, mechanical stimulation of an AT2-like cell resulted in a high incidence of propagated [Ca2+]i changes to AT1-like cells or AT2-like cells. In the presence of apyrase, mechanical stimulation of AT1-like cells was not affected, however, mechanical stimulation of AT2-like cells resulted in a reduced propagation of [Ca2+]i changes to AT2-like cells, and a significant decrease in propagation to AT1-like cells. In the presence of gap26 mechanical stimulation of AT1-like cells resulted in a significantly reduced propagation of [Ca2+]i change to both AT1-like cells and AT2-like cells. Mechanical stimulation of an AT2-like cell in the presence of gap26 resulted in a reduced propagation of [Ca2+]i change in AT1-like cells and AT2-like cells. When apyrase and gap26 were applied together, Ca2+ communication was significantly reduced in all cases.

 
In homogenous cultures of AT2-like cells, intercellular Ca2+ waves can propagate through a paracrine mechanism that is sensitive to the nucleotidase, apyrase (11). Homogenous cultures of AT1-like cells also respond to wound-induced release of nucleotides to coordinate [Ca2+]i changes among neighboring cells (23). In heterocellular cultures both AT1-like and AT2-like cells retained their ability to respond to 10 µM ATP or 10 µM UTP by increasing their [Ca2+]i, and this response was inhibited by 50 U/ml apyrase (data not shown). To determine whether the observed changes in mechanically-induced Ca2+ signaling occurred by a paracrine signaling or gap junctional pathway in heterocellular cultures, Ca2+ communication was studied in the presence of inhibitors to each signaling pathway, apyrase was used to eliminate signaling via extracellular nucleotide/purinergic receptors, and gap26 was used to block signaling via gap junctions (Table 1). In the experiments with apyrase, mechanical stimulation of a single AT1-like cell resulted in high incidence of Ca2+ communication to AT1-like and to AT2-like cells (Figure 5). In contrast, mechanical stimulation of an AT2-like cell significantly reduced Ca2+ communication to neighboring AT1-like cells. Apyrase only partially reduced Ca2+ signaling from AT2-like cells to neighboring AT2-like cells. When heterocellular cultures were exposed to gap26, mechanical stimulation of AT1-like cells resulted in a significant reduction in Ca2+ communication to both neighboring AT1-like and to AT2- like cells. Conversely, gap26 did not significantly reduce Ca2+ waves after mechanical stimulation of AT2-like cells. To determine whether coordinated [Ca2+]i changes between cells was due to intercellular communication, both apyrase and gap26 were used together. Under these conditions, mechanical stimulation of AT1-like cells resulted in minimal Ca2+ communication to both AT1-like and AT2-like cells. Mechanical stimulation of AT2-like cells in the presence of apyrase and gap26 also significantly reduced propagated changes in [Ca2+]i to AT1-like and AT2-like cells. In each case, removal of the inhibitors by incubating cells in fresh HBSS resulted in Ca2+ waves that resembled controls within 15 min. The inhibitor studies combine to show that coordinated [Ca2+]i changes in heterocellular cultures of alveolar epithelial cells are cell type specific and are facilitated by either/both paracrine nucleotide and/or gap junctional signaling.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cells in the alveolar epithelium coordinate their cellular physiology by using a variety of intercellular signaling pathways including paracrine (extracellular) and gap junctional (direct) communication mechanisms. In the context of gap junctional signaling pathway, second messengers and small metabolite transfer between cells can be regulated by gap junction protein (connexin) expression, trafficking, or post-translational modification (43). Alveolar epithelial cells show great variability in the connexin expression patterns that may in part be due to changes in extracellular matrix or neighboring cells (17). In this study, connexin profiles of the alveolar epithelium in rat lung sections (in situ) and in heterocellular cultures (in vitro) were determined by immunocytochemistry and shown to be similar between in situ and in vitro models for the alveolar epithelium. Functional dye coupling and coordinated changes in [Ca2+]i among neighboring cells were investigated in the heterocellular culture model. Dye coupling and Ca2+ communication were observed between and among both AT1-like and AT2-like cells. However, differences in coupling and communication suggest that stimulated cells regulated the extent of intercellular communication to neighboring cells.

Studies on connexin expression in lung tissue and cell cultures have shown that alveolar epithelial cells contain a complex pattern of protein expression that include Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 (7, 11, 19–24). The positive staining for Cx26 and Cx40 in this report are the first to show these connexin isoforms in rat alveolar sections, whereas the staining for Cx32, Cx43, and Cx46 are in agreement with that seen in previous work (19). Although there are reports of Cx37 expression in lung tissue from mouse (44) and rat (20), their absence from alveolar lung sections in this report agrees with the suggestion that Cx37 is limited in alveolar epithelial cells (45) and likely expressed in endothelial cells in the lung (46). Within the heterocellular cultures, where AT1-like and AT2-like cells were more easily discernable by morphology, each cell phenotype displayed a distinct expression pattern of connexin isoforms. The staining patterns seen with Cx32, Cx43, and Cx46 are in agreement with the alveolar section staining from this and other studies (19), where it is suggested that both AT1 and AT2 cells express these isoforms. The association of Cx40 staining to areas that contain AT2-like cells, the dispersed patterns of Cx26, and the lack of Cx37 staining in heterocellular cultures are also in agreement with what can be determined from alveolar sections in this study.

Connexin isoform expression in homogenous alveolar epithelial cell cultures of AT1-like or AT2-like cells are different from the heterocellular culture expression patterns. In AT2 cell isolations that were allowed to differentiate toward AT1-like cells, there is a consistent pattern of plasma membrane staining for Cx40 (23) and Cx43 (7, 22, 23, 26, 47), a juxtanuclear staining pattern for Cx46, and limited expression of Cx26 and Cx32 isoforms (23). Cultured AT2 cells express a different connexin profile with prominent plasma membrane expression of Cx26, consistent expression of Cx46, and differential expression of Cx32 and Cx40 that is dependent on keratinocyte growth factor (7, 11). The noted differences between connexin expression patterns in monocellular cultures from previous studies and those seen in the heterocellular cultures or alveolar slices from this study supports a plasticity of connexin expression that can be influenced by neighboring cell phenotype or cell matrix. Additionally, the similarity between connexin isoform expression in heterocellular cultures and in situ alveolar slices from this study suggests an improved model for studying cell communication in vitro.

Although the functional significance of multiple connexin isoform expression in alveolar cells is unknown, it is thought generally that differential connexin expression regulates channel properties and subsequent intercellular signaling events (17). In support of this, it has been shown using paired Xenopus oocytes that differential transfer of dye or small metabolites can be due to changes in connexin isoform makeup of the functional gap junction, the amount of functional gap junctions between the cells, or the molecular properties of the molecule being transferred between cells (48, 49). Homogenous cultures of alveolar cells with AT1-like or AT2-like phenotype were shown to be coupled after injection of LY (11, 24). A technique in which freshly isolated AT2 cells are laid down on top of 6-d-old cultures of alveolar epithelial cells showed dye coupling of both LY and the fluorescent tracer dye calcein (MW = 622; six negative charges) between these two cell types (19, 22). In an attempt to isolate the connexin isoforms that can participate in this type of coupling, transfected HeLa cells that expressed either Cx43 or Cx32 were loaded with calcein and used as donor cells for 6-d-old cultures of pulmonary alveolar cells (19). The HeLa cells expressing Cx43 (but not Cx32) were dye coupled to the alveolar epithelial cells within 5 h of cell–cell contact. These studies (19) showed that coupling can occur between Cx43 expressing cells and pulmonary alveolar cells within hours of contact, but they did not address communication in cells that have established full functional junctions. In the present report, microinjection of tracer dyes LY or A350 were used to study functional coupling in 7-d-old cultures of AT1-like and AT2-like cells that expressed tight junctions (14). Heterocellular cultures were functionally coupled with LY or A350, although LY transfer from AT2-like to AT1-like cells was significantly reduced when compared with transfer from AT1-like to AT1-like, from AT1-like to AT2-like or, from AT2-like to AT2-like cells (Figures 3 and 4; Table 1). The dye coupling properties and the distinct connexin profiles of AT1 and AT2 cells could be a consequence of the different makeup of gap junctions connecting these cells.

By using several inhibitors with a variety of efficacies and potencies to uncouple gap junctional signaling (41, 50), further information on communication mechanisms and specificities of alveolar cells was obtained. Connexin mimetic peptides were used because of their ability to quickly and reversibly block gap junctional coupling in airway epithelial cells (11, 23, 40), and their potential to selectively block gap junctions made of specific connexin isoforms (41, 42, 51). The peptide mimetic, gap27, reduced LY and A350 dye coupling with the exception of transfer from AT1-like cells to AT2-like cells. Independent of the dye, gap27 did not significantly reduce dye coupling when AT2-like cells were the donor and AT1-like cells were recipients. To clarify specificity of block, a second mimetic peptide, gap26, was used and shown to uncouple all dye transfer, independent of the donor/recipient cells. Our data points to an unidirectional bias in dye transfer. There is precedence for connexin mimetic peptide-specific block of dye coupling via gap junctions made from different connexin isoforms. In COS-7 cells, a monkey fibroblast cell line that expresses Cx43 as the sole connexin isoform, gap27 effectively uncoupled LY dye transfer, whereas the corresponding peptide to Cx40 (Table 2) was ineffective (42). A comparison of connexin isoform sequences in the regions corresponding to gap26 and gap27 of Cx43 for connexins detected in AT1-like and AT2-like cells shows significant differences (Table 2), which may lead to a change in efficacy of inhibition. The differential uncoupling effect of gap27 when compared with gap26 and the apparent unidirectional bias of dye transfer between AT1-like and AT2-like cells are consistent with different connexin isoforms in the gap junctions of the phenotypically distinct cell pairs. A similar situation in vivo may allow for selective metabolite and second messenger coupling between cells in the alveolar epithelium. Unidirectionally-biased dye transfer has been reported between neuronal/astrocyte cell pairs (52, 53), although these cells have also been shown to be bidirectionally-coupled (54).

The mechanisms of action of connexin mimetic peptides are not understood (41). The ability for several different connexin mimetic peptides to block dye transfer and/or second messenger signaling with slightly different efficacies in alveolar and tracheal airway cell cultures suggests that the inhibition is more than a nonspecific interaction (11, 23, 40). This is further supported by the lack of inhibition of gap junctional communication by connexin mimetic peptides derived from intracellular loop sequences, or derived by scrambling the amino acid sequences of the gap26 or gap27 peptides (11, 23, 40; data not shown). The rapid reversibility of block seen in tracheal airway epithelial cultures argues against internalization of the peptides or a sole mechanism of action by blocking connexon/connexon pairing (40). Further studies are required to define the inhibitory mechanisms of connexin mimetic peptides and how this may help to elucidate individual connexin isoforms that are important in functional coupling between and among AT1 and AT2 cell phenotypes.

Cellular communication of physiologic signals in the alveolar epithelium can coordinate tissue function. For example, communication of [Ca2+]i changes from AT1 cells to AT2 cells in situ resulted in release of surfactant from AT2 cells (18). Alternatively, the contribution of AT1 cells to the surface area combined with the expression of sodium pumps and water channels suggests that communication of second messenger molecules from AT2 to AT1 cells may contribute to an altered air/liquid interface in the alveoli. Because dye coupling experiments do not directly address the coordination of the transfer of physiologically significant molecules and ions, the mechanisms underpinning the propagation of Ca2+ waves from mechanically stimulated to neighboring cells were studied. Using cell culture models, it has been shown that coordination of [Ca2+]i among alveolar epithelial cells can occur through at least two mechanisms: (i) second messenger coupling via gap junction channels, or (ii) paracrine stimulation of purinergic receptors through ATP/UTP release (reviewed in Ref. 17). Using mechanical stimulation to initiate communication has shown that unwounded AT1-like cells in homogenous cultures did not release ATP or UTP, rather, [Ca2+]i changes were propagated via gap junctions (23). Conversely, AT2-like cells in homogenous cultures communicated [Ca2+]i changes via nucleotide-mediated paracrine signaling (11). To determine the pathway for intercellular Ca2+ waves between phenotypically distinct alveolar cell types, we mechanically stimulated single cells in heterocellular cultures and applied inhibitors to differentiate between the two major signaling pathways. As in homogenous cultures, mechanical stimulation of either cell type resulted in Ca2+ communication to neighboring cells. Also similar to the mechanism elucidated from homogenous cultures, mechanical stimulation of AT1-like cells communicated increases in [Ca2+]i to neighboring AT1-like or AT2-like cells using gap junctions. In contrast, mechanical stimulation of AT2-like cells in heterocellular culture was more complex. Stimulated AT2-like cells communicated increases in [Ca2+]i to neighboring AT1-like and AT2-like cells mainly via an apyrase-sensitive mechanism, suggesting that ATP or UTP were extracellular mediators of communication. This mechanism of transfer is consistent with that seen in homogenous cultures of AT2-like cells (11). However, stimulated AT2-like cells in heterocellular culture also communicated Ca2+ changes to neighboring AT2-like cells via gap junctions (Figure 5). This dual mechanism of transfer further suggests regulation of physiologic signal transfer via gap junctions can be affected by extracellular factors such as neighboring cells not participating in the gap junctional communication or altered extracellular matrix.

The reasons for this shift in mechanisms used for Ca2+ communication between alveolar cell phenotypes are not clear, but the anatomy of the alveolar epithelium may provide clues. AT1 cells are situated side by side, and in contact with AT2 cells in normal alveolar epithelium, an arrangement that favors efficient gap junctional signaling. In contrast, AT2 cells in the normal alveolar epithelium are not in direct contact and are more likely to communicate Ca2+ changes to each other via a paracrine pathway. AT2 cells also proliferate, migrate, and differentiate to reform the epithelium after lung repair and during this process, AT2 cells are in contact with each other, allowing gap junctional signaling to be an efficient way of coordinating second messenger signaling. Further, spatiotemporal differences in [Ca2+]i changes and the availability of specific signaling pathways for Ca2+ in the target cell can change the physiologic outcome of the Ca2+ signal (55). For example, secretion kinetics in AT2 cells are altered by spatiotemporal changes in Ca2+ signaling generated by different Ca2+ agonists (56). Therefore, changes in Ca2+ communication mechanisms may influence the Ca2+ signaling patterns and subsequent physiologic outcomes in the target cell. Further study on cell communication and physiologic outcome will help to elucidate roles for different connexin isoform expression and changes in mechanisms of Ca2+ communication in the alveolar epithelium.


   Acknowledgments
 
The authors thank Dr. R. Heinzen for use of microinjection facility, Drs. Z. Zhang and P. R. Wade for suggestions on lung sectioning and staining, and Dr. P. E. M Martin for helpful discussion with connexins. This work was supported in part by a Research Travel Award from the Burroughs Wellcome Fund (B.E.I.) and NIH grants HL64636, HL64039, RR15553 (S.B.), HL03609 (R.L.L.). G.J.S. is an L. Floyd Clarke scholar.

Received in original form November 30, 2002

Received in final form May 9, 2003


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 Results
 Discussion
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A. S. Patel, D. Reigada, C. H. Mitchell, S. R. Bates, S. S. Margulies, and M. Koval
Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation
Am J Physiol Lung Cell Mol Physiol, September 1, 2005; 289(3): L489 - L496.
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T. J. King and P. D. Lampe
The Gap Junction Protein Connexin32 Is a Mouse Lung Tumor Suppressor
Cancer Res., October 15, 2004; 64(20): 7191 - 7196.
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 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Boitano, Z. Safdar, D. G. Welsh, J. Bhattacharya, and M. Koval
Cell-cell interactions in regulating lung function
Am J Physiol Lung Cell Mol Physiol, September 1, 2004; 287(3): L455 - L459.
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