1 Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071-3166; and 2 Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom
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ABSTRACT |
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Inter- and extracellular-mediated changes in intracellular Ca2+ concentration ([Ca2+]i) can ensure coordinated tissue function in the lung. Cultured rat alveolar epithelial cells (AECs) have been shown to respond to secretagogues with increases in [Ca2+]i and have been shown to be gap junctionally coupled. However, communication of [Ca2+]i changes in AECs is not well defined. Monolayers of AECs were mechanically perturbed and monitored for [Ca2+]i changes. Perturbation of AECs was administered by a glass probe to either mechanically stimulate or mechanically wound individual cells. Both approaches induced a change in [Ca2+]i in the stimulated cell that was propagated to neighboring cells (Ca2+ waves). A connexin mimetic peptide shown to uncouple gap junctions eliminated Ca2+ waves in mechanically stimulated cells but had no effect on mechanically wounded cells. In contrast, apyrase, an enzyme that effectively removes ATP from the extracellular milieu, had no effect on mechanically stimulated cells but severely restricted mechanically wounded Ca2+ wave propagation. We conclude that AECs have the ability to communicate coordinated Ca2+ changes using both gap junctions and extracellular ATP.
calcium; cell communication; connexins; adenosine 5'-triphosphate
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INTRODUCTION |
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PULMONARY ALVEOLAR EPITHELIAL CELLS (AECs) line the lung and provide an impermeable barrier against the external environment. The primary constituents of the alveolar epithelium are alveolar type I (ATI) and alveolar type II (ATII) cells. ATI cells are thought to provide a pathway for gas exchange, whereas ATII cells have been shown to be more physiologically diverse. ATII cells provide critical surfactant (e.g., Ref. 54) and extracellular matrix (ECM) protein (e.g., Ref. 19) secretions, participate in H2O and ion transport to regulate fluid composition of the lung (40), and play a crucial role in response to injury, disease, and infection within the lung (16, 40, 42). The ability of ATII cells to act as progenitors of ATI cells (reviewed in Ref. 52) is vitally important for the lung response to injury. After acute alveolar epithelial injury, ATII cells can migrate into a denuded area of lung epithelium, differentiate to an ATI phenotype to restore the epithelial layer, and prevent fibrosis (20, 56). A similar transition occurs in primary cultures of ATII cells after several days in culture on a collagen matrix where ATII cells can lose specific cell markers and take on a more flattened, or ATI type, morphology (15). Dysfunction of AECs in vivo, such as seen after severe cases of acute respiratory distress syndrome, infection, or injury, can have serious effects on lung physiology, including reduced O2 transfer, surfactant release, or fibrosis. Although intercellular communication via gap junctions or extracellular signaling may provide for such coordination, there is little known concerning the specific cellular mechanisms that allow for AECs to coordinate epithelial function in the lung.
Changes in intracellular Ca2+ concentration ([Ca2+]i) are a common theme in initiating cellular processes in a variety of cells (3, 4), including those of the lung epithelium (for reviews, see Refs. 11, 37, 41, 48). Despite the growing abundance of in vitro modeled Ca2+ events involving ATII cells, there is little known concerning if and how coordination of Ca2+ signaling occurs to aid in the tissue response in vitro or in vivo. Because of the ability to spatiotemporally control the initiation of cellular signaling, micropipette perturbation of cultured cells has emerged as an attractive technique to induce coordinated changes in [Ca2+]i for the study of intercellular communication (reviewed in Ref. 48). Two major pathways for Ca2+ communication have been proposed for mechanically induced Ca2+ waves, gap junctional intercellular communication (GJIC) and the release of extracellular signaling molecules, most commonly ATP; these pathways need not be mutually exclusive (12, 14, 21, 30, 51). Recent reports (1, 25, 28, 31, 33) support a role for gap junctions in cell-cell signaling in AECs. However, these reports elucidate considerable differences in the expression of gap junction proteins (connexins) in AEC cultures. The mixed results from these studies and the aforementioned potential for ATP-dependent signaling emphasize that the mechanisms of communication in AECs may not be straightforward, and current studies to assess the significance of the varied gap junction expression and their function in AECs are inadequate to model intercellular signaling in AECs.
In this study, connexin expression patterns in 7-day cultured AECs were identified by immunocytochemistry. Additionally, localized mechanical force was used on single cells to initiate two distinct types of intercellular Ca2+ signaling (Ca2+ waves) in these cultures. When single cells are mechanically perturbed without compromising the plasma membrane (mechanical stimulation), Ca2+ waves are propagated through gap junctions. However, if mechanical perturbation results in the release of cytosolic contents (mechanical wounding), Ca2+ waves are primarily propagated via extracellular diffusion of ATP. These redundant pathways for intercellular Ca2+ communication and the varied effects of Ca2+ signaling suggest an important role for Ca2+ waves in the alveolar epithelium.
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MATERIALS AND METHODS |
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Chemicals. Hank's balanced salt solution (HBSS), Dulbecco's modified Eagle's medium and Ham's F-12 medium in a 1:1 ratio, bovine serum albumin (BSA), trypsin inhibitor, penicillin, streptomycin, and amphotericin were purchased from GIBCO BRL (Life Technologies, Grand Island, NY). Fura 2 and fura 2-acetoxymethyl ester (fura 2-AM) were purchased from Calbiochem (La Jolla, CA). Elastase was purchased from Worthington Biochemical. Gap 27 peptide derived from rat cDNA of connexin (Cx) 43 to include amino acids 204-214 (SRPTEKTIFII) from the second extracellular loop was purchased from Genosys (Cambridge, UK) or Severn Biotech (Worcester, UK). Antibodies were raised against the conserved peptide sequences from individual connexins and have been shown to react to connexins from several mammalian species. Antibodies generated to Cx26 were raised in rabbit against a human peptide sequence. Antibodies to Cx32 and Cx43 were raised in rabbit against rat peptide sequences. Antibodies to Cx40 (rabbit anti-mouse) and Cx46 (rabbit anti-rat) were purchased from Alpha Diagnostics International (San Antonio, TX). Secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO) and were of the highest grade available.
Cell cultures. Primary ATII cells were harvested by modified methods previously described (6). Male Sprague-Dawley rats weighing 250-300 g were killed by peritoneal pentobarbital sodium injection. The lungs were initially 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), 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) and 40 mg/ml of elastase. The lungs were minced in a trypsin inhibitor solution (100 mg of BSA, 10 mg of trypsin inhibitor, 10 mg of DNase, 40 µl of 100 mM EDTA stock, and 10 ml of solution I per rat) and panned on IgG plates for 1 h at 37°C. The cells were plated on a fibronectin (1 µg/µl)-type I collagen matrix over a 15-mm glass coverslip at a density of 1.8 × 106 cells/ml. The cells were grown in Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and amphotericin B at 37°C in a 5% CO2 atmosphere for 7 days. After day 4, the culture medium was changed every day. Morphology of the ATII cells was confirmed with a 1.0% tannic acid stain as previously described (38). Cell viability was determined with trypan blue (5%) exclusion and was consistently >90%.
Application of inhibitors. Monolayer cells were incubated with a Gap 27 peptide [130 µM (0.25 mg/ml) in HBSS] for 35-45 min. Apyrase (50 U/ml in HBSS) was applied 2 min before ATP application.
Immunocytochemistry. The cells were washed with cold HBSS and incubated with cold 50:50 acetone-methanol for 3 min. The cells were washed with phosphate-buffered saline (PBS; 8 mM Na2HPO4, 128 mM NaCl, 2 mM KCl, and 10 mM HEPES, pH 7.4), incubated for 20 min with antibody dilution solution [0.25% gelatin, 3.0% BSA, 0.05% Tween 20, and 0.2% NaN3 in Tris-buffered saline (150 mM NaCl and 15 mM Tris, pH 7.4)], and incubated with the appropriate primary connexin antibody diluted 1:100 and applied for 1 h at 37° C. The coverslips were washed with PBS and antibody dilution solution and incubated in a diluted 1:250 anti-rabbit IgG-FITC conjugate for 1 h at 37°C. The coverslips were thoroughly washed with PBS and deionized water, mounted, and viewed on a Leica TSD-4D confocal laser scanning microscope.
Ca2+ imaging. The cells were loaded with fura 2 by a 75-min incubation with 5 µM fura 2-AM in 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 additionally buffered with 25 mM HEPES, pH 7.4). The cells were washed for 20 min in HBSS, mounted on an inverted Olympus IX70 microscope, and observed with a ×40, 1.35-numerical aperture oil-immersion objective. Fura 2 fluorescence was observed after excitation was alternated at 340 and 380 nm by a 75-W xenon lamp linked to a Delta Ram illuminator [Photon Technologies (PTI)]. Images of emitted fluorescence above 510 nm were recorded by an intensified charge-coupled device camera (PTI) and simultaneously displayed on a 21-inch vivitron color monitor. The imaging system was under software control (ImageMaster, PTI) on an IBM clone computer. Calculation of [Ca2+]i was by published equations (24). A positive response in [Ca2+]i was determined when [Ca2+]i increased over 150 nM, a three- to fourfold increase over resting [Ca2+]i. All cells exhibiting a positive change in [Ca2+]i were counted as communicating cells. Thus a Ca2+ wave of 1 cell represents a [Ca2+]i change only in the stimulated cell.
Mechanical stimulation and wounding. A glass micropipette (tip diameter ~1 µm) under piezoelectric control was positioned with a hydraulic micromanipulator and deflected downward for 150 ms to deform an individual AEC. If the membrane of the deformed cell was not broken by the micropipette, it was considered to be mechanically stimulated; if the membrane of the deformed cell was broken by the micropipette, resulting in dye release, it was considered to be mechanically wounded.
Statistics. Two-sample Student's t-tests were used in all data comparisons. Results are averages ± SD.
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RESULTS |
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Morphology of cultured ATII cells.
ATII cells were isolated as described in MATERIALS AND
METHODS. The cells were visually checked for morphology and
stained for lamellar bodies, an ATII cell marker, to determine cell
type in culture. At isolation, ATII cells comprised >99% of the cells (325 of 325 cells counted from 2 isolations stained positively for
lamellar bodies; data not shown). On day 2 of culture, ATII cells had a typical cuboidal shape ~5-10 µm across, with
>98% of the cells staining positive for lamellar bodies (120 of 122 cells counted from 2 isolations; Fig.
1A). On day 7 of
culture, the cells were more flattened in shape, were ~20-25
µm across, and displayed a reduced number of lamellar bodies, with
29% of the cells staining positive for the ATII cell marker (100 of
345 cells counted from 2 isolations had at least one lamellar body; Fig. 1B). Because 7-day-old cultures displayed
characteristics of both ATII and ATI cells, they are best described as
AECs.
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Connexin localization in AECs.
We used immunocytochemistry and specific antibodies generated to Cx26,
Cx32, Cx40, Cx43, and Cx46 to study the expression of connexin proteins
in 7-day-old AEC cultures. Immunocytochemistry with antibodies
generated to Cx40 (Fig. 2A)
and Cx43 (Fig. 2B) resulted in a punctate staining pattern
of AECs at the plasma membranes, indicative of these connexins directly
participating in cell-cell communication. Staining with antibodies to
Cx46 (Fig. 2C) also showed distinct punctate staining.
However, this staining was localized to the juxtanuclear region of the
AECs. Immunocytochemistry of AECs with antibodies generated to Cx32
(Fig. 2D), and Cx26 (Fig. 2E) resulted in some
cellular localization near the juxtanuclear region that was
significantly higher than background (Fig. 2F). However,
cell surface punctate staining with antibodies to Cx26 and Cx32 was
severely reduced compared with results with antibodies generated to
Cx40, Cx43, and Cx46.
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AEC response to ATP.
ATP has been used as an effector molecule to raise
[Ca2+]i in lung epithelium and other tissues
and has been suggested as an extracellular messenger for coordinated
Ca2+ communication. To determine whether 7-day-old AECs
responded to ATP, we monitored [Ca2+]i in
monolayer cultures of AECs after exposure to 10 µM ATP. Application
of ATP resulted in a significant rise in
[Ca2+]i in AECs (Fig.
3, A-D). All
cells in the field of view responded uniformly with an increase in
[Ca2+]i by 60 s and a return to baseline
levels of [Ca2+]i by 120 s. To
manipulate ATP response, 50 U/ml of apyrase were added to 10 µM ATP
immediately before bath application (Fig. 3, E and
F). Apyrase effectively eliminated the
[Ca2+]i response to 10 µM ATP in AECs.
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Mechanical stimulation and Ca2+
communication in AECs.
To determine whether AECs were capable of communicating
[Ca2+]i changes,
[Ca2+]i was monitored in a field of cells
during and after a central cell was briefly stimulated with a glass
micropipette. Mechanical stimulation (where the plasma membrane was not
damaged by the micropipette) resulted in a Ca2+ wave that
averaged slightly over 4 cells (n = 17; Figs.
4, A-D, and
5A). All cells
returned to baseline [Ca2+]i within 120 s. The gap junction-inhibiting peptide Gap 27 (5, 13, 53)
and the ATPase apyrase were used to determine the pathway of
Ca2+ communication after mechanical stimulation. After a
30-min incubation with Gap 27, mechanical stimulation resulted in an
increase in [Ca2+]i that was largely
restricted to the stimulated cell (n = 7; Figs. 4,
E-H, and 5A). This inhibition of
communication was a significant reduction from Gap 27-free mechanical
stimulation (P < 0.001). A 15-min washout of Gap 27 allowed for a return to normal-sized Ca2+ waves on
mechanical stimulation (Fig. 5A). Similar experiments with
apyrase did not significantly reduce Ca2+ wave propagation
in response to mechanical stimulation (n = 17; Figs. 4,
I-L, and 5A).
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Mechanical wounding and Ca2+
communication in AECs.
To determine whether cell communication between AECs could occur by
alternative signaling mechanisms, a glass micropipette was introduced
to a single AEC such that the plasma membrane was compromised and the
intracellular contents were free to mix with the extracellular milieu,
and the [Ca2+]i in AEC cultures was measured.
After such mechanical wounding, AECs displayed Ca2+ waves
that averaged greater than six cells (n = 17; Figs.
5B and 6,
A-D). As above, Gap 27 and apyrase were used
to determine the mechanism of Ca2+ wave propagation.
A 30- to 45-min incubation with Gap 27 did not significantly
reduce the size of the mechanical wound-induced Ca2+ wave
(Figs. 5B and 6, E-H). However,
when apyrase was applied to the AEC monolayer for 2 min or more,
mechanical wound-induced Ca2+ waves were restricted to the
stimulated cell and one or two neighboring cells (n = 11; Figs. 5B and 6, I-L). This
was a significant reduction in wound-induced Ca2+ waves
measured in apyrase-free cultures (P < 0.001; Fig.
5B). Washout of apyrase for 5 min allowed for a return of
mechanical wound-induced Ca2+ waves to near control values
(n = 8; Fig. 5B).
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DISCUSSION |
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Cellular communication pathways are required for coordinated tissue function, including that of the alveolar epithelium. In this report, we show that 7-day-old cultures of AECs (from purified ATII cells grown on a fibronectin-collagen matrix) express connexin proteins required for GJIC. Through the use of localized mechanical perturbation of a single cell, it is further demonstrated that AECs can coordinate [Ca2+]i changes among neighboring cells (Ca2+ waves) through two distinct pathways, namely, GJIC and extracellular diffusion of ATP released from the mechanically perturbed cell. The redundancy of pathways for propagation of Ca2+ waves suggests an important function for this type of communication in AECs.
Gap junctions are constructed of connexin proteins, of which ~20 major isoforms are identified in mammals. Expression of connexins and their intracellular trafficking and assembly into gap junctions at cell contact areas is an important indicator that GJIC may occur. In this work, antibodies generated to Cx26, Cx32, Cx40, Cx43, and Cx46 were used to study connexin expression in 7-day-old AECs. It was shown that AECs have punctate expression of Cx40 and Cx43 at the plasma membrane in addition to Cx46 at the juxtanuclear region. Any combination of these connexins could contribute to the GJIC of Ca2+ waves detected after mechanical stimulation (Figs. 4 and 5). Several recent reports (e.g., Refs. 1, 25, 33) have used a variety of techniques to elucidate connexin expression patterns in rat AECs, mostly focusing on 0- to 4-day-old primary cultures of ATII cells. With Northern and immunoblot analyses in rat ATII cells to detect Cx26, Cx32, Cx43, and Cx45, day 0 isolations consisted of Cx26 and Cx32 expression, whereas Cx43 and Cx45 were not detected (33). By day 3 of culture, however, Cx32 and Cx45 were absent, Cx26 was reduced in expression, and Cx43 was the most commonly expressed connexin. Significantly, by day 6 in culture, the immunoblot analysis of Cx43 showed a reduced protein expression, but the Northern blot analysis reported continued high expression of Cx43 mRNA. In a separate study (1), rat ATII cells were analyzed for 13 different connexins at isolation with RT-PCR; these cells displayed Cx26, Cx30.3, Cx32, Cx37, Cx43, and Cx46 on day 0. In the same study, connexin expression was followed over time with total mRNA assays for the recognized connexins. On day 4 of culture, total mRNA for Cx43 and Cx46 increased and for Cx26 and Cx32 decreased, whereas Cx30.3 and Cx37 remained constant. If the culture medium was changed by reducing the fetal bovine serum supplement and adding keratinocyte growth factor to increase ATII cell morphology (e.g., Ref. 7), the patterns of connexin expression were altered, with Cx32 and Cx30.3 significantly increased in mRNA levels (1). Although our studies show Cx43 and Cx46 expression in day 7 cultures of AECs, neither of the previous studies looked for Cx40, a predominant connexin in 7-day-old AECs (Fig. 2). It has been shown that ECM components can influence ATII cell structure and function (for review, see Ref. 19), and, likewise, ATII cells can influence the ECM (e.g., Ref. 18). A recent study (32) in keratinocytes has also linked connexin expression and gap junctional communication with changes in the ECM. Because the results discussed above include ATII cell isolations grown on various ECM components, discrepancies in the findings of connexin expression may be linked to the ECM. However, it should be noted that the ECM also likely changes in vivo during wound healing as the cell types that migrate over the ECM can contribute to changes in the ECM. Because it has become increasingly clear that the connexin makeup of gap junctions allows for selectivity of molecule transfer (e.g., Ref. 23), the varied connexin expression displayed in AEC cultures over time may be an important part of cellular communication in vivo. That is, the expression of connexins during ATII cell differentiation may allow for transfer of select signaling molecules to coordinate distinct physiological processes in the alveolar epithelium. We further speculate that a trigger for the initial changes in [Ca2+]i communicated after the pathophysiology and subsequent release of cytosolic constituents from damaged cells in vivo could occur in the absence of gap junctional signaling.
It has long been postulated that mechanical forces play an important
role in both developing (e.g., Ref. 2) and mature (e.g.,
Refs. 39, 46) lungs in vivo. In vitro,
mechanical stretch of ATII cells grown on Silastic membranes has been
shown to induce cellular signaling in AECs (e.g., Ref.
55). In this study, we focused mechanical forces onto a
single cell and measured the ability of that cell to communicate
responses to mechanical force. Mechanical perturbation allows distinct
spatiotemporal control to initiate intercellular signaling
communication of Ca2+ waves through two independent
mechanisms dependent on the health status of the stimulated cell. In
other in vitro cell systems, including ovine lens cells
(14), liver cells (21), osteoblasts (30), and pancreatic cells (9), a duality in
mechanisms for mechanically induced Ca2+ waves also exists.
However, of these systems, only the ovine lens cells were shown to
require wounding for ATP-dependent signaling. In a previous study
(29) with AECs where [Ca2+]i
responses to mechanical wounding were measured, wounding was induced by
dragging an 18-gauge needle across ATII cell cultures to induce
Ca2+ waves encompassing hundreds of cells; it was
postulated that these Ca2+ waves were dependent on an
extracellular messenger presumed to be ATP. Our observations with
Ca2+ waves induced by mechanical wounding of a single cell
sensitive to apyrase confirm that nucleotide release from wounded cells can result in a Ca2+ wave. GJIC in AECs has also been
analyzed with dye transfer (1, 25, 33) and mechanical
stimulation-induced Ca2+ communication (33).
In these studies, traditional gap junctional inhibitors such as
18-glycyrrhetinic acid (25) and octanol (33) could be used to uncouple dye transfer (25,
33) or Ca2+ signaling (33) via GJIC in
day 2-4 isolated ATII cells. In the present
study using Gap 27, a benign, nonpenetrant, and reversible GJIC peptide
inhibitor (5, 13, 43, 53), mechanical stimulation-induced Ca2+ communication in 7-day-old AECs is reversibly blocked.
In contrast to previous results, the present study emphasizes the
importance of AEC communication in two ways: 1) the
communication of physiologically significant molecules such as the
second messenger Ca2+ and 2) the redundancy of
mechanisms for Ca2+ wave propagation to ensure proper
communication in AECs.
Ca2+ is an important signaling molecule in AECs. AEC [Ca2+]i can be raised by changes in pH (22), mechanical stretch (55), insulin (36), endothelin (50), ATP (17, 44), and heavy metal application (34) among other means. Changes in [Ca2+]i have distinct physiological effects on AECs. Such changes have been shown to be involved in membrane reorganization (8); secretion of surfactant (27, 44, 45, 47, 49, 55), arachidonic acid metabolites (10), and ECM molecules (18); regulation of channels (35); differentiation of ATII to ATI cells (26); and repair of the epithelial barrier (29). The communication of Ca2+ waves either by GJIC (as in mechanical stimulation) or by extracellular signaling molecules (as in mechanical wounding) can provide a mechanism for the coordination of Ca2+-induced physiology (or pathophysiology) in the AECs that line the lung alveolus. How AECs interact in vivo in relation to Ca2+ wave communication remains to be studied, but speculation in relation to the above-mentioned effects that [Ca2+]i changes have on AECs is intriguing. Coordination of surfactant or other secretions from ATII cells, differentiation from ATII to ATI phenotype, migration of AECs to cover a denuded epithelial area, or regulation of ion channel activity within the alveolar epithelium are just some of the possible physiological outcomes of Ca2+ waves in AECs. In further studies, the use of available mechanisms for coordinated changes in [Ca2+]i and subsequent physiological outcomes may be elucidated to better understand the function of the alveolar epithelium in vivo.
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ACKNOWLEDGEMENTS |
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We thank A. B. Narahara for surgical expertise and S. T. Stoddard, C. E. Olsen, N. A. Groathouse, A. Omsland, C. Seger, and G. J. Seedorf for cell culture and helpful comments on imaging sequences. We also thank R. L. Lubman for helpful advice in the isolation and culture of alveolar type II cells.
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FOOTNOTES |
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This work was supported by Research Grant RG-0420N from the American Lung Association, National Heart, Lung, and Blood Institute Grant HL-64636 (both to S. Boitano), Programme Grant G-9305117 from the Medical Research Council (to W. H. Evans), and a Basic Research Grant from the University of Wyoming College of Arts and Sciences (to S. Boitano).
B. E. Isakson is an L. Floyd Clarke Scholar.
Address for reprint requests and other correspondence: S. Boitano, Dept. of Zoology and Physiology, PO Box 3166, Univ. of Wyoming, Laramie, WY 82071-3166 (E-mail: sboitano{at}uwyo.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.
Received 17 May 2000; accepted in final form 25 July 2000.
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