Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Evidence of absorptive or secretory ion transport in different
respiratory regions of the mouse was sought by assessing the regional
distribution of -,
-, and
-epithelial sodium channel (ENaC; Na+ absorptive), cystic fibrosis transmembrane
conductor regulator (CFTR), and
Na+-K+-2Cl
cotransporter mRNAs.
High levels of ENaC subunit expression were found in nasal surface
epithelium and gland ducts. CFTR was expressed in both superficial
nasal respiratory epithelium and glands. These results are consistent
with basal amiloride-sensitive Na+ absorption and
cAMP-dependent Cl
secretion in murine nasal epithelia.
Expression of all three ENaC subunits increased progressively from
trachea to terminal bronchioles. Intermediate levels of CFTR and
cotransporter expression in bronchial epithelium diminished in
bronchioles. The low abundance of CFTR mRNA throughout murine pulmonary
epithelium is consistent with functional data that attributes
Cl
secretion predominantly to an alternative
Cl
channel.
-ENaC as the only mRNA found in all
regions of airway epithelia is consistent with the
-subunit as
requisite for Na+ absorption, and the increased expression
of
-,
-, and
-ENaC in distal airways suggests a greater
absorptive capability in this region.
cystic fibrosis transmembrane conductor regulator; epithelial sodium channel; sodium potassium-chloride cotransporter; nasal
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INTRODUCTION |
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AIRWAY SURFACE LIQUID lines all airways of the murine lung and is transported via ciliary beating and viscous liquid flow-dependent mechanisms from distal to proximal regions (25). This liquid may be secreted by alveolar or bronchiolar epithelia. If liquid is secreted by alveoli, then liquid reabsorption may be the predominant function of the most distal bronchioles because the relatively large alveolar surface area converges to the relatively smaller surface area of the distal airways. On the other hand, small airways may serve as the primary region for secretion of ions and liquid that move up airway surfaces.
Liquid secretion and absorption by epithelia are driven by active
transport of solutes (mainly electrolytes) across the epithelial barrier. Solute transport creates local concentration gradients that
result in liquid movement by osmosis through or around the epithelial
cells (11). An amiloride-sensitive epithelial sodium channel (ENaC) is found in the apical membrane of pulmonary epithelia. This channel in series with a basolateral
Na+-K+-ATPase constitutes the path for active
Na+ flow that drives counterion (Cl) and
water absorption across the epithelium. The rate-limiting step for
Na+ transport typically is at the level of ENaC, and in
airway epithelia, cystic fibrosis transmembrane conductor regulator
(CFTR) can regulate ENaC (36). Conversely, liquid
secretion is mediated by entry of Cl
from the
interstitium into the cell through a
Na+-K+-2Cl
cotransporter in the
basolateral membrane, followed by exit across the apical membrane
through Cl
channels such as CFTR. The rate-limiting
mechanism for sustained Cl
secretion in several epithelia
is the cotransporter (31).
To understand how the epithelium in each region of the pulmonary tree effects salt and water transport, the expression of transport and channel proteins must be linked to functional measurements from the same region. Although ion and water transport by large-airway epithelia has been studied in vivo (18, 21) and in vitro (5, 20, 25, 34), evaluation of distal lung epithelia has been hampered by the small size and architecture of the bronchioles and alveoli. Immunolocalization of ion channel and transporter proteins is also difficult because these proteins are not abundant on the surface of the epithelia. However, differences in the relative abundance of ion translocation molecules along the respiratory tract of rats (14, 26) and humans (8) have been inferred from different levels of mRNA expression.
Gene-targeted mice can demonstrate the functional consequences of the
loss of a specific protein in vivo or in excised tissue or cells
removed from these animals. Currently, gene-targeting approaches are
restricted to the mouse, a species for which there is little
information about the distribution of transport and channel proteins in
the lung. Consequently, we mapped the relative abundance of the ENaC
subunits (,
, and
), CFTR, and cotransporter within different
regions of the murine respiratory tract (alveolar, bronchiolar,
bronchial, tracheal, and nasal). We compared these data with the
absorptive and secretory functions published for the proximal airway
regions (nasal, tracheal, and bronchial) to determine whether mRNA
distribution could predict ion transport in distal lung epithelia.
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METHODS |
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Tissue Preparation
Nasal septum, trachea, and lungs were excised from 7- to 8-wk-old mice of wild-type, mixed (BL6, 129, DBA) lineage. Mice were killed with CO2, and the chest cavity was opened to expose the lungs. A long polypropylene pipette tip was inserted into the trachea through the larynx, and 3-5 ml of optimal cutting temperature (OCT) compound embedding medium (Sakura Finetek; Torrance, CA) diluted 1:1 with phosphate-buffered saline were injected to inflate the lung. Then the lung and trachea were removed from the cavity, embedded in OCT compound in a rectangular polypropylene embedding mold, and frozen with dry ice. The nasal tissue was isolated by removing the overlaying cartilage, resecting a lateral section, and embedding the section in OCT compound. Frozen blocks of tissue were sectioned (7 µm thick) and mounted on glass slides. Tissue sections were fixed in 4% paraformaldehyde for 60 min, dehydrated, and stored atIn Situ Hybridization
Frozen sections were equilibrated at room temperature, digested with proteinase K, and hybridized with the 35S-labeled UTP probe (200-800 ng of plasmid with theProbe Preparation
Antisense and sense probes were prepared from mouse ENaC cDNAs (Data Analysis
Exposure times for each hybridized mRNA probe were determined from the time that was required to obtain an antisense signal of
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RESULTS |
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Nasal
Superficial epithelium.
Expression of all mRNAs was found in the superficial epithelium (Fig.
1 and Table 1). Expression of -ENaC,
-ENaC, and cotransporter was homogeneously distributed in a layer
lining the apical side of the epithelium, whereas the distribution of
CFTR and
-ENaC was localized to a subpopulation of cells. CFTR and
-ENaC silver grains were found clustered around 10-20% of the
respiratory epithelial cell nuclei in the sections of nasal respiratory
epithelium as shown in Fig. 1. This percentage of CFTR and
-ENaC
expressing cells was localized to gland duct openings (J. Harkema,
personal communication). In olfactory epithelium, the highest level of CFTR was found in 35-50% of the cells populating the apical
surface (Fig. 1), a percentage that probably corresponds to the
distribution of sustentacular or supporting cells (28).
The homogeneous distribution of ENaC subunits and cotransporter
expression in olfactory epithelium (data shown for
-ENaC only) was
similar to that found in respiratory epithelium. The level of
expression of all mRNAs appeared higher in the olfactory than in the
respiratory epithelium, perhaps due to the greater number of cell
layers that comprise the olfactory epithelium. The highest levels of
cotransporter and CFTR expression throughout the entire respiratory
tract were found in the nasal epithelium (Fig.
2 and Table 1).
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Glands.
There was a low level of expression for -ENaC and
-ENaC in all
gland acini (Fig. 2 and Table 1). A high level of cotransporter expression was found in some serous acini of the lateral nasal glands.
CFTR was highly expressed in subepithelial serous gland acini of the
septum (Fig. 2). In contrast, expression of
-ENaC was not detected
in acini but was noted at high levels in gland duct openings of the
lateral nasal gland (data not shown). The level of
-ENaC,
-ENaC,
and CFTR expression in the gland ducts was comparable to that in the
epithelium (Fig. 2). Cotransporter could not be detected in duct structures.
Trachea
The only mRNA seen after the set exposure times (see METHODS) throughout the superficial epithelia of the trachea with antisense signal (silver grain density, 2.3 ± 0.3 silver grains/nucleus) above background was
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Bronchi and Bronchioles
All mRNAs were expressed in airway epithelia, but the distribution of cotransporter and CFTR mRNAs was different from the pattern of expression for the ENaC subunits. The levels of all ENaC subunits increased progressively from proximal (lobar bronchus) to distal airway (terminal bronchiole). This increase was most pronounced forAlveoli
Intermediate levels of
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Extrapulmonary Thoracic Tissues
Esophagus. There was an intermediate level of expression of all ENaC subunits in esophageal epithelium and muscularis mucosa (data not shown). Expression of CFTR or cotransporter was not detected in esophagus (data not shown).
Thyroid gland.
Gland tissue attached to the sides of trachea was characterized by
moderate levels of expression of - and
-ENaC and low levels of
-ENaC and cotransporter (Fig. 3).
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DISCUSSION |
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Even though mice are a convenient animal model for studying the
gene-dependent pathogenesis of diseases such as cystic fibrosis, the
gene products may play relatively different roles in the mouse than in
humans. For example, cystic fibrosis lung disease is not prevalent in
CF-deficient mice (35). Functional studies demonstrate amiloride-inhibitable Na+ absorption and cAMP-dependent
Cl secretion (16, 17) in mouse
nasal epithelia that mimic the pattern of transport in human airways.
In contrast, these are not dominant mechanisms of ion transport in
lower airway (trachea) epithelia. For example, mouse airways appear to
secrete Cl
by a mechanism that does not require a
cAMP-dependent Cl
channel (CFTR) (17). These
functional studies demonstrate the diversity of ion transport processes
among species, and indeed airway regions, and that mouse nasal may be a
better model than mouse tracheal epithelium for ion transport by human
airway epithelia.
The present study had two goals: 1) to define the correlation between mRNA expression and the functional evidence of salt and water transport in well-studied regions (proximal airways) of mouse pulmonary epithelium and 2) if a positive correlation was determined, then to predict dominant ion transport (based on mRNA expression) in regions of the epithelium for which there are little or no data. One caveat to these predictions is that mRNA expression does not prove that there is a comparable or universally proportional level of functional protein in that region.
Nasal
We focused initially on the pattern of ion transport mRNA expression in the nasal region because ion transport by murine airways has been most thoroughly characterized in this region and because ion transport mechanisms in the mouse nasal and human large-airway epithelia are similar (16). Coexpression of mRNAs encoding all ion translocating proteins in the nasal superficial epithelium suggests that this epithelium can modulate surface liquid by a balance of absorption and secretion. Coexpression of the three ENaC subunits suggests Na+ absorption capability, whereas the combination of CFTR and cotransporter expression is consistent with ClBecause gland acini are secretory in function, we used this site to test the correlation between expression of CFTR and the cotransporter (but not ENaC mRNAs) and secretion. A relatively high level of both CFTR and cotransporter mRNA expression in certain glandular regions of mouse nose parallels secretory function reported for gland acinar cells (22). ENaC mRNA expression was relatively low and similar to studies of gland acini of other species (8, 14). Expression of these subunits in acinar cells may support the secretory process by "recycling" Na+ across the apical membrane (38).
Submucosal gland ducts modify volume and/or composition of the
primary secretion from acinar cells in some species. High levels of
-,
-, and
-ENaC expression in mouse nasal gland duct are consistent with the capacity to absorb Na+ from the ductal
lumen as in sweat duct (32). CFTR expression in the ductal
epithelium could regulate Na+ absorption and thereby
suggest two roles for CFTR function. If absorption in this region, like
absorption by the sweat duct, is mediated by an epithelium with minimal
paracellular ion flow, then the CFTR Cl
conductance could
limit the rate of net NaCl transport (30). Alternatively,
CFTR could modulate absorptive rates via regulation of the activity of
ENaC as it does in airway epithelial cells (23).
The high levels of expression of all mRNAs encoding transport proteins found in olfactory epithelium indicates this epithelium contributes to luminal salt and water balance in the nose. Olfactory epithelium consists of neuroepithelial sensory cells and supporting cells (sustentacular) that are thought to regulate the salt concentration surrounding the neurosensory ciliated cells (15). Several ion transport elements have been functionally and biochemically characterized in sustentacular cells: 1) a voltage-independent, large potassium conductance, 2) amiloride-sensitive Na+ channels, and 3) Na+-K+-ATPase (13, 37). Our mRNA data confirm the presence of ENaC and also suggest CFTR channel and cotransporter function in sustentacular cells. These data support the idea that sustentacular cells maintain ion gradients in the liquid layer that surrounds the olfactory epithelium.
Trachea
Compared with nasal epithelium, the portion of total ion transport contributed by amiloride-sensitive Na+ or cAMP-dependent ClWhen regions of the tracheal surface that cover the cartilage vs.
posterior membrane were compared, - and
-ENaC were coexpressed with
-ENaC only in the membranous region. Because studies of oocyte
expression and ENaC subunit-deficient mice demonstrate increased
Na+ currents and Na+ absorption when
- and
-ENaC are coexpressed with
-ENaC, a regional difference in ENaC
expression suggests that the magnitude of Na+ transport in
functional studies depends on the fraction of total luminal surface
exposed in the Ussing chamber that is contributed by the posterior membrane.
In general, the levels of mRNA expression for all transport proteins
were lower in trachea than in nose and lower airways. Different
regional patterns of ENaC subunit expression are not unique to mouse
airways. The relative level of mRNA for each subunit varies
significantly among regions of the rat and human respiratory tract as
well (8, 14, 26). The absence of
CFTR expression in trachea is consistent with immunocytochemical data
(41) and with Cl transport studies that
suggest Cl
conductance in this region is mediated via a
Ca2+-activated Cl
channel rather than through
CFTR. The absence of cotransporter in this region also suggests that
the entry step mediating Cl
secretion is different from
that in nasal submucosal glands. The lack of mRNA expression for any of
these transport proteins in the tracheal submucosal glands suggests
that these glands are functionally distinct from nasal glands.
Bronchial and Bronchiolar Expression
Functional studies of murine bronchi and bronchioles have been hampered by the small size of these structures. Accordingly, our studies of mRNA expression provide the first information about ion translocation mechanisms in these regions of the mouse lung. We can summarize the data as two different trends in mRNA expression. First, there are relatively high levels of all three ENaC subunits expressed throughout surface epithelia of the distal airways (bronchus-terminal bronchiole). The increase in the level of ENaC expression,Second, the relatively low level of CFTR expression in the bronchial
surface epithelium is even lower in the bronchiolar epithelium. The
lower levels of CFTR expression throughout the mouse lung compared with
human supports the basic observation that CF knockout mice do not
develop lung disease and that another mechanism for Cl
transport, probably a Ca2+-dependent Cl
channel, accounts for ion and liquid secretion in mouse lung. The
existence of an alternative Cl
secretory pathway(s)
complicates interpretations that link reduced capacity to secrete
Cl
with diminished CFTR expression in the more distal
airway regions.
Alveolar Region
Alveolar epithelium is mainly composed of two cell types: type I cells that occupy the majority of the alveolar surface area and type II cells that typically reside in alveolar corners and comprise ~5% of the surface area (29). ENaC expression was detected only in alveolar corners, consistent with expression in type II cells. The inference that these cells are capable of Na+ transport is supported by functional studies of cultured type II cells from adult rats and rabbits by Mason et al. (24) and others. The relatively low level of CFTR in alveolar corners together with a lack of detectable cotransporter is consistent with the absence of CFTR-mediated ClIf the functional and expression data from type II cells and distal bronchioles are representative of Na+ absorptive physiology, then the site of pulmonary surface liquid secretion remains unknown. There are at least three plausible sources. First, if active ion transport drives liquid onto pulmonary surfaces, then the liquid that moves up airway surfaces forms on bronchiolar surfaces, and a secretory process that is not identified from our mRNA expression data is responsible. Second, type I cells, which cover the majority of the alveolar surface, secrete ions and liquid. Because the surface occupied by type I cells is very large, mRNA (and expressed protein) could be widely dispersed and virtually undetectable. However, the aggregate function of these transport proteins could secrete enough liquid to cover the alveolar (and bronchiolar) surface. Third, liquid could form on alveolar surfaces in response to passive forces. If alveolar epithelia are highly water permeable (12), then it is possible that the air-liquid interface may generate small negative pressures in the surfactant hypophase sufficient to drive liquid flow. Although this process moves liquid onto the alveolar surface, it does not deliver salt across an epithelium with low NaCl permeability (33). Clearly, the shortcomings of each of these hypotheses point to the need to further characterize the net salt and water transport by the intact alveolar surface and the role of type I pneumocytes in this process.
In summary, patterns of epithelial CFTR, cotransporter, and ENaC
mRNA distribution and studies of epithelial ion transport suggest that
mouse nasal epithelia more closely resemble human large-airway
epithelia than mouse trachea. Mouse lung is anatomically and
functionally distinct from human in that 1) there are very few submucosal glands, 2) there are no respiratory
bronchioles, 3) the majority of Cl conductance
moves through a channel other than CFTR, and 4) CF-deficient mice
hyperabsorb Na+ across nasal but not airway epithelia. We
found a low abundance of mRNA for CFTR throughout the pulmonary
epithelium that is consistent with this functional pattern. In
contrast, a progressive increase in ENaC expression from proximal to
distal airways (terminal bronchioles) suggests a greater absorptive
capability for bronchioles.
-ENaC was the only mRNA found in all
regions of lung epithelia (nasal, airways, and alveolar). This is
consistent with the
-subunit as requisite for Na+ absorption.
Finally, both "transport" molecule (i.e., ion channels as well as transporter) expression and the limited functional studies with cultured cells or even with excised tissue fail to account for the origin of surface liquid on distal pulmonary epithelia (airway and alveolar). Most studies predict or describe absorption, but the fact remains that surface liquid must be secreted within this region. We suggest three hypothetic processes that could result in liquid secretion by distal pulmonary epithelia and point the way to future studies.
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ACKNOWLEDGEMENTS |
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We thank Drs. John Gatzy and Barbara Grubb for careful and thorough reviews of this manuscript. Thanks also to Kim Burns and Tracy Bartolotta for histochemical preparation of the tissues, to Dr. Jack Harkema for identification of nasal anatomic structures, and to Dr. David Parsons for assistance with identification of nasal epithelia.
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FOOTNOTES |
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This work was supported by the Cystic Fibrosis Foundation and National Institute of Health Grants HL-34322 and HL-60280.
Address for reprint requests and other correspondence: L. G. Rochelle, CF/Pulmonary Research Center, CB No. 7248, 928 MEJB UNC-Chapel Hill, Chapel Hill, NC 27599 (E-mail: lrochell{at}med.unc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 22 July 1999; accepted in final form 3 February 2000.
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