Distribution of ion transport mRNAs throughout murine nose and lung

Lori G. Rochelle, Dong Chen Li, Helen Ye, Eddie Lee, Colleen R. Talbot, and Richard C. Boucher

Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Evidence of absorptive or secretory ion transport in different respiratory regions of the mouse was sought by assessing the regional distribution of alpha -, beta -, and gamma -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. alpha -ENaC as the only mRNA found in all regions of airway epithelia is consistent with the alpha -subunit as requisite for Na+ absorption, and the increased expression of alpha -, beta -, and gamma -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta , and gamma ), 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -20°C in an airtight box.

In 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 the alpha -, beta -, or gamma -ENaC or cotransporter insert) or 35S-labeled UTP plus CTP probe (500-800 ng of plasmid with the CFTR insert) as described previously (8). The final specific activity of the labeled riboprobe in the hybridization solution was ~15,000 dpm/µl. Tissue sections were washed with decreasing stringency solutions of NaCl and sodium citrate, coated with NBT-2 autoradiography emulsion (Kodak), and exposed for 6-7 days for ENaC subunits, 22-26 days for CFTR, and 20-23 days for cotransporter at 4°C before being developed and stained with hematoxylin and eosin. Serial sections from three to four animals were hybridized with sense and antisense probes for each cDNA.

Probe Preparation

Antisense and sense probes were prepared from mouse ENaC cDNAs (alpha -665 bp 1263-1927, beta -449 bp 63-511, and gamma -687 bp 583-1270) by RT-PCR with primer oligomers that were 30 bp in length. These PCR products were cloned into the PCR II TA vector (Invitrogen) and linearized with EcoR V or Hind III. CFTR375 (bp 1346-1706) cDNA was cloned into pBluescript KS and linearized with BamH I or Kpn I for antisense-sense pairs. Cotransporter cDNA (bp 2507- 3133) (10) was cloned into PCR 2.1 vector (Invitrogen) and linearized with EcoR V and Hind III.

Data Analysis

Exposure times for each hybridized mRNA probe were determined from the time that was required to obtain an antisense signal of >= 5 silver grains/nucleus in the region of highest expression. For alpha -, beta -, and gamma -ENaC, this region was the distal bronchioles. For cotransporter and CFTR, the region was the nasal olfactory epithelium. For Tables 1 and 2, the level of expression in the region of highest expression was set as "+++," and other regions were set at relatively lower levels of expression based on silver grain density. Silver grain density was quantified as the number of silver grains per nucleus in antisense minus the number per nucleus in the paired sense tissue section. The silver grains in six random regions (2 regions in 3 mice, and region area 30-40 nuclei) of each airway generation (trachea, bronchus, and bronchiole) were examined under dark field using the ×50 objective of a Leitz Orthoplan microscope for both antisense and sense probed tissue sections. Silver grain density in bronchial epithelium was compared with that in either trachea or bronchiole by Student's t-test.

                              
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Table 1.   Mouse nose relative regional intensities


                              
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Table 2.   Mouse airway relative regional intensities


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nasal

Superficial epithelium. Expression of all mRNAs was found in the superficial epithelium (Fig. 1 and Table 1). Expression of alpha -ENaC, beta -ENaC, and cotransporter was homogeneously distributed in a layer lining the apical side of the epithelium, whereas the distribution of CFTR and gamma -ENaC was localized to a subpopulation of cells. CFTR and gamma -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 gamma -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 alpha -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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Fig. 1.   In situ hybridization of alpha -epithelial sodium channel (alpha -ENaC), beta -ENaC, gamma -ENaC, cystic fibrosis transmembrane conductor regulator (CFTR), and cotransporter (CoT) in nasal respiratory and olfactory (olf) epithelia. Arrows point to gland duct openings. Bright-field [hematoxylin and eosin (HE)] image of each frozen section is shown on the left. Dark-field images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each probe. Magnification ×400.



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Fig. 2.   In situ hybridization of alpha -ENaC, beta -ENaC, gamma -ENaC, CFTR, and CoT in nasal septum or turbinate. Discernible tissues are the superficial epithelium that overlays both sides, subepithelial gland acini (A) and ducts (arrows), and cartilage in the middle of the section. The bottom row is the lateral nasal gland with a high level of CoT expression in the acini. Bright-field (HE) image of each frozen section is shown on the left. Dark-field images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each probe. Magnification ×100, bottom row ×200.

Glands. There was a low level of expression for alpha -ENaC and beta -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 gamma -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 alpha -ENaC, beta -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 alpha -ENaC (Fig. 3 and Table 2). The level of expression of alpha -ENaC was less (Fig. 3 and Table 2) than that in more distal airways (Fig. 4 and Table 2). beta -ENaC and gamma -ENaC were also detected but only in the epithelium that overlays the membranous portion of the trachea (data not shown) and not in the epithelium that covers the cartilage. Consequently, the three ENaC subunits were expressed only in the superficial epithelium of the membranous portion. None of the mRNAs was detected in the submucosal glands that are clustered at the proximal end of the trachea (Fig. 3).


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Fig. 3.   In situ hybridization of alpha -ENaC, beta -ENaC, gamma -ENaC, CFTR, and CoT in trachea. The superficial epithelium lines the lumen. The proximal end, where submucosal glands (SMG) are located, is at the left edge of each panel. T, thyroid gland. Bright-field (HE) image of each frozen section is shown on the left. Dark-field images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each probe. Magnification ×18.



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Fig. 4.   In situ hybridization of alpha -ENaC, beta -ENaC, gamma -ENaC, CFTR, and CoT in bronchi (left half of each panel) through bronchioles (right half of each panel). Bright-field (HE) image of each frozen section is shown on the left. Dark-field images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each probe. Magnification ×18.

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 for beta -ENaC (Fig. 4 and Table 2) where the silver grain density increased from 4.6 ± 0.7 in bronchus to 8.4 ± 1.0 silver grains/nucleus in bronchioles (P = 0.01). The silver grain density for gamma -ENaC in bronchus was 4.0 ± 0.9 and 4.3 ± 0.8 in bronchioles. The increase in alpha -ENaC expression from bronchus (5.6 ± 1.1) to bronchioles (6.6 ± 1.0) was not as pronounced as the increase from trachea (2.3 ± 0.8) to bronchus (P = 0.02). The highest level of expression for all ENaC subunits was found in the terminal bronchioles. In contrast, the intermediate level of cotransporter and low level of CFTR expression found in the surface epithelium of bronchus extended to terminal bronchioles but decreased progressively in a proximal to distal orientation.

Alveoli

Intermediate levels of alpha -, beta -, and gamma -ENaC subunit expression along with a low level of CFTR and cotransporter were found in the corners of the alveoli, presumably in type II cells (Fig. 5).


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Fig. 5.   In situ hybridization of alpha -ENaC, beta -ENaC, gamma -ENaC, CFTR, and CoT in bronchioles and alveoli. Bright-field (HE) image of each frozen section is shown on the left. Dark-field images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each probe. Magnification ×100

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 alpha - and beta -ENaC and low levels of gamma -ENaC and cotransporter (Fig. 3).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cl- secretion. These results are compatible with functional data that describe a relatively large basal amiloride-sensitive Na+ absorption as well as the capacity for induction of cAMP-dependent Cl- secretion in freshly excised mouse nasal tissue (17).

Because 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 alpha -, beta -, and gamma -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 Cl- flow across the mouse trachea is variable. For example, the amiloride-sensitive component of the short-circuit current (net ion flow) across excised trachea varies between 30 and 70% (16). The pattern of mRNA expression may help explain this variability. Only alpha -ENaC expression was detected over most of the surface epithelium. Relationships between Na+ transport rates and ENaC subunit expression have been explored in oocyte studies (9, 27) and studies of ENaC subunit-deficient mice (4, 19). A low rate of Na+ transport can be mediated by expression of the alpha -subunit alone. The hypothesis that mouse airway epithelia require expression of alpha -ENaC to absorb Na+ is supported by the observation that newborn gene-targeted mice without alpha -ENaC survive for less than 48 h because liquid is not removed from their lungs (19). The rate of absorption is decreased in gamma -ENaC-deficient mice (4).

When regions of the tracheal surface that cover the cartilage vs. posterior membrane were compared, beta - and gamma -ENaC were coexpressed with alpha -ENaC only in the membranous region. Because studies of oocyte expression and ENaC subunit-deficient mice demonstrate increased Na+ currents and Na+ absorption when beta - and gamma -ENaC are coexpressed with alpha -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, alpha -, beta -, and gamma -ENaC, from bronchial to terminal bronchiolar regions, suggests that the relative capacity for Na+ absorption increases in the most distal airway (terminal bronchiolar) regions. Our data from the bronchial epithelium are consistent with ion transport studies of bronchial epithelia from several species (human, canine, ovine, and porcine) (3, 5-7) that showed Na+ absorption but little or no basal or inducible Cl- secretion. Our data are also consistent with findings from fewer ion transport studies of freshly excised bronchioles from rat (2), pig (3), and sheep (1) that absorption dominates the NaCl flow across this barrier. Ion transport data from cultured bronchiolar preparations are mixed. Whereas cultured human airways exhibit secretion (40), cultured rabbit bronchiolar cells (Clara cells) are dominated by Na+ absorption (39). Which of these systems best models in vivo conditions is unknown, and species differences have not been thoroughly investigated.

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 Cl- secretion in most type II cell cultures. Consequently, our hypothesis that links patterns of transport protein expression with function is supported by the evidence for type II cells from other species.

If 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. alpha -ENaC was the only mRNA found in all regions of lung epithelia (nasal, airways, and alveolar). This is consistent with the alpha -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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