Aquaporins in complex tissues. II. Subcellular distribution in
respiratory and glandular tissues of rat
Søren
Nielsen1,
Landon S.
King2,3,
Birgitte Mønster
Christensen1, and
Peter
Agre2,4
1 Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK 8000 Aarhus C,
Denmark; and 2 Department of
Medicine, 3 Division of
Pulmonary and Critical Care Medicine, and
4 Department of Biological
Chemistry, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205-2185
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ABSTRACT |
The molecular pathways for fluid transport in pulmonary, oral,
and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of four
aquaporins in the respiratory tract and glandular epithelia, where they
reside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is present
in apical and basolateral membranes of bronchial, tracheal, and
nasopharyngeal vascular endothelium and fibroblasts. AQP5 is localized
to the apical plasma membrane of type I pneumocytes and the apical
plasma membranes of secretory epithelium in upper airway and salivary
glands. In contrast, AQP3 is present in basal cells of tracheal and
nasopharyngeal epithelium and is abundant in basolateral membranes of
surface epithelial cells of nasal conchus. AQP4 resides in basolateral
membranes of columnar cells of bronchial, tracheal, and nasopharyngeal
epithelium; in nasal conchus AQP4 is restricted to basolateral
membranes of a subset of intra- and subepithelial glands. These sites
of expression suggest that transalveolar water movement, modulation of
airway surface liquid, air humidification, and generation of
nasopharyngeal secretions involve a coordinated network of aquaporin
water channels.
immunocytochemistry; immunoelectron microscopy; alveolus; nasopharyngeal epithelium; secretory glands
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INTRODUCTION |
DISCOVERY OF THE AQUAPORIN family of water channel
proteins has provided a molecular explanation for rapid water movements across the plasma membranes of cells (2). Aquaporin-1 (AQP1) is a
constitutively activated water channel (27) and has been identified in
multiple tissues, including red blood cells, renal proximal tubules,
and capillary endothelium (7, 25, 26, 29), whereas AQP2 is restricted
to renal collecting duct, where it is regulated by vasopressin (6, 14,
22). AQP3 is present in the basolateral plasma membranes of the renal
collecting duct (9, 13). AQP4 is highly abundant in perivascular glial
cells and in ependymal cells (13, 23). The
Aqp5 cDNA was isolated from rat
salivary gland (28).
Movements of water in the distal lung, airways, oral cavity, and
nasopharynx are normally involved in clearance of alveolar fluid,
airway humidification, and generation of oral and nasal secretions,
whereas abnormal movements of water are found in clinical disorders
such as pulmonary edema, freshwater drownings, cystic fibrosis, asthma,
allergic rhinitis, and Sjögren's syndrome (17). Aquaporins may
participate in these processes, but understanding of these proteins in
lung, airways, oral cavity, and nasopharynx is incomplete.
Developmental immunoblots have defined the expression patterns of
aquaporins in lung and nasopharynx; however, cellular and subcellular
sites of expression are incompletely resolved. AQP1 is present in the
pulmonary visceral pleura and in peribronchiolar capillary endothelium,
where expression is induced by corticosteroids, whereas AQP1 is
sparsely present in alveolar capillary endothelium (18); AQP3 and AQP4
have been described in basolateral membranes of tracheal epithelia
(13); AQP5 mRNA is strongly expressed in lung, but its cellular location is undefined (28). Moreover, no
aquaporin has been defined in alveolar epithelium (17), and expression
of aquaporins has yet not been assessed in nasopharynx.
In an accompanying study (19), affinity-purified rabbit antibodies to
AQP1, AQP3, AQP4, and AQP5 were shown to react specifically with 28- to
30-kDa polypeptides on immunoblots of respiratory and glandular
tissues. Here we use the same antibodies for a comprehensive analysis
of the cellular and subcellular distribution of AQP1, AQP3, AQP4, and
AQP5 in the lung and upper airways to provide insight into the roles of
aquaporins in the complex physiology of the respiratory tract and
glandular tissues.
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MATERIALS AND METHODS |
Antibodies.
Anti-peptide antibody specific for AQP5 was prepared and characterized
as described in the accompanying study (19). The synthetic peptide
(NH2-CEPEEDWEDHREERKKTIELTAH-COOH)
corresponding to the COOH-terminus of AQP5 was cross-linked to keyhole
limpet hemocyanin and injected into New Zealand White rabbits
(Lofstrand Laboratories, Gaithersburg, MD). Polyclonal anti-AQP5
immunoglobulin G (IgG) was affinity purified from serum, using
Sulfolink coupling gel (Pierce) conjugated with 2-4 mg of the
synthetic peptide. As a negative labeling control, anti-AQP5 was
preincubated with a 100-fold excess of the immunizing peptide at
4°C for 24 h. Polyclonal, affinity-purified rabbit antibodies to
AQP1 (anti-AQP1) that react with the 4-kDa COOH-terminal domain of the
protein were previously described (29). Affinity-purified
peptide-derived antibodies against AQP3 (kindly provided by Dr. Mark
Knepper, National Institutes of Health, Bethesda, MD) and AQP4 have
previously been characterized in detail (9, 23, 30).
Preparation of tissues for immunocytochemistry.
Adult male Wistar rats were anesthetized, heparinized, and
perfusion-fixed through the right atrium with 4 or 8% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Tissues were postfixed for
2 h in the same fixative, infiltrated for 30 min with 2.3 M sucrose
containing 2% paraformaldehyde, mounted on holders, and rapidly frozen
in liquid N2. These blocks were
used for cryosectioning or freeze substitution with embedding in
Lowicryl HM20. With use of an automatic freeze substitution system
(AFS, Reichert, Vienna, Austria), samples were sequentially
equilibrated over 3 days in 0.5% uranyl acetate in methanol, at
temperatures gradually decreasing from
80°C to
70°C, and then rinsed in pure methanol for 24 h at
70°C to
45°C (21, 22, 25). At
45°C,
the samples were serially infiltrated with Lowicryl HM20 and methanol
1:1, then 2:1, and finally with pure Lowicryl HM20, before ultraviolet
polymerization for 2 days at
45°C and 2 days at 0°C.
Immunohistochemistry.
For light microscopy, cryosections were placed on gelatin-coated glass
slides and processed as described (22, 24, 25). After preincubation
with phosphate-buffered saline containing 1% bovine serum albumin or
0.1% skimmed milk and 0.05 M glycine, the sections were incubated with
anti-aquaporin antibodies overnight at 4°C. The use of the
affinity-purified antibodies against AQP1, AQP3, AQP4, and AQP5 for
immunocytochemistry has previously been described (9, 22-26, 30).
The following concentrations (in µg/ml) were used: 0.1-0.2
anti-AQP1, 0.5 anti-AQP3, 1 anti-AQP4, and 0.5-2 anti-AQP5. The
labeling was visualized with peroxidase-conjugated secondary antibody
(P448, 1:100, DAKO A/S, Glostrup, Denmark). Sections were
counterstained with Meier counterstain.
Immunoelectron microscopy.
Ultrathin Lowicryl sections (60-80 nm) or ultrathin cryosections
(80 nm) were obtained with a Reichert Ultracut FSC ultracryomicrotome. The sections were incubated with affinity-purified anti-AQP5 or anti-AQP3 (described in
Antibodies) and
labeling was visualized with goat anti-rabbit IgG conjugated to 10-nm
colloidal gold particles (GAR.EM10, BioCell Research Laboratories,
Cardiff, UK). The sections were stained with uranyl acetate for
10 min or with uranyl acetate for 10 min followed by lead citrate for
15 s, and examined in a Philips CM100 electron microscope.
Immunolabeling controls.
The following controls confirmed specificity of light and electron
microscopic studies: 1) incubation
with protein A-purified preimmune rabbit IgG,
2) adsorption of anti-AQP5 (0.1 µg/ml) with the immunizing AQP5 peptide (10 µg/ml), and
3) incubations without primary
antibody or without primary and secondary antibody.
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RESULTS |
Cellular and subcellular distribution of aquaporins in distal lung.
Immunocytochemical studies performed on 0.9-µm cryosections of distal
lung using affinity-purified antibodies revealed that AQP5 is expressed
in alveolar type I pneumocytes (Fig. 1,
A and B). AQP5 was not
detected in type II pneumocytes, vascular endothelium, or interstitial
cell types (Fig. 1, A and
B), and specificity was demonstrated
by nonimmune control (Fig. 1C).
Immunoelectron microscopy also demonstrated that AQP5 is present only
in the apical membrane of type I pneumocytes (Fig.
2, A and
D), with no expression in type II
cells (Fig. 2C) or capillary
endothelium (Fig. 2, A and
D). Specificity was established by
nonimmune controls (Fig. 2, B and
E), and AQP1, AQP3, and AQP4 were
not detected in alveolar epithelium of rat (not shown).

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Fig. 1.
Immunocytochemical localization of aquaporins in rat lung.
Cryosections (0.9 µm) of distal rat lung
(A-C),
bronchus (D,
F, and
H), and trachea
(E,
G, and
I) were labeled with
affinity-purified antibodies and visualized with peroxidase-conjugated
secondary antibody. A and
B: type I pneumocytes exhibit
aquaporin-5 (AQP5) immunolabeling (arrows), whereas endothelial cells
of capillaries (A, arrowheads) and
venules (B, arrowheads), myocytes, and
fibroblasts are unlabeled. ×1,000.
C: immunolabeling control using
nonimmune immunoglobulin G (IgG) in place of anti-AQP5 reveals absence
of labeling. ×480. D: section of
bronchus reveals no labeling with anti-AQP3. ×480.
E: basal cells of ciliated,
pseudostratified tracheal epithelium exhibit strong immunolabeling for
AQP3 (arrows). Except for occasional sites that most likely represent
sectioning artifact, basolateral and apical membrane of surface
epithelial cells (arrowhead) are unlabeled. ×1,000. Sections of
bronchus (F) and trachea
(G) demonstrate expression of AQP4
on lateral (arrows) and basal plasma membranes of surface cells in
pseudostratified columnar epithelium but are distinct from those
labeled with anti-AQP3 (E); apical
membranes are unlabeled. ×1,000. Bronchus
(H) and trachea
(I) exhibit no labeling with
anti-AQP5. ×480.
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Fig. 2.
Immunoelectron microscopic localization of AQP5 in distal rat
lung. Ultrathin cryosections
(A-C)
or ultrathin Lowicryl sections (D and
E) were labeled with
affinity-purified anti-AQP5 and visualized with goat anti-rabbit IgG
conjugated to 10-nm colloidal gold particles. A: type I pneumocytes exhibit labeling
with anti-AQP5 in apical plasma membranes (arrows); basolateral plasma
membranes are unlabeled (arrowheads). Capillary endothelial cell (EN)
is unlabeled. ×60,000. EP, epithelial cells.
B: control with nonimmune IgG reveals
absence of immunolabeling. Arrowheads, basolateral plasma membranes.
×60,000. C: section of type II
pneumocyte with lamellar granule (G) reveals negligible labeling with
anti-AQP5. ×60,000. D: Lowicryl
section of type I pneumocyte showing anti-AQP5 labeling of apical
plasma membrane (arrows) with no labeling of basolateral membrane
(arrowheads) or adjacent endothelial cell. ×60,000.
E: control using nonimmune IgG reveals
absence of immunolabeling. ×60,000. Note that majority of
immunogold particles in A and
D appear to overlie apical plasma membrane, whereas only a single immunogold particle is found in C and most likely represents
background staining.
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Cellular and subcellular distribution of aquaporins in bronchus and
trachea.
AQP4 is present in basolateral membranes of bronchial epithelium (Fig.
1F) and tracheal epithelium (Fig.
1G), whereas AQP3 is restricted to
only the tracheal epithelium (Fig. 1,
D and
E), consistent with the distribution
found by immunoblotting (19). The AQP3 and AQP4 antibodies label
distinctly different cells in the ciliated, pseudostratified tracheal
epithelium. AQP3 appears only in the basal cells of the tracheal
epithelium, where it is heavily expressed (Fig.
1E). In contrast, AQP4 is present in
the basolateral plasma membranes of the ciliated columnar cells that reach the epithelial surface and is not expressed in basal cells (Fig.
1G). This labeling pattern was
confirmed by immunoelectron microscopy revealing abundant AQP3 in basal
cells (Fig. 3,
A and B) but absence of AQP3 in surface
epithelial cells. AQP5 was not detected in either bronchial or tracheal
surface epithelium (Fig. 1, H and
I).

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Fig. 3.
Immunoelectron microscopic localization of AQP3 in rat trachea.
Ultrathin Lowicryl sections were labeled with affinity-purified anti-AQP3 and visualized with goat anti-rabbit IgG conjugated to 10-nm
colloidal gold particles. A: plasma
membranes of basal cells (BC) in tracheal epithelium exhibit extensive
AQP3 immunolabeling (arrows), but surface epithelial cells (SE)
displayed no labeling. Immunolabeling of basal cells is notable in
plasma membrane processes interdigitating with unlabeled processes of
surface epithelial cells. B: basal
plasma membrane of basal epithelial cells facing basement membrane also
display heavy anti-AQP3 labeling (arrows). Surface epithelial cells are
unlabeled. ×75,000.
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Cellular and subcellular distribution of aquaporins in nasopharynx
and conchus.
Complex patterns of aquaporin distribution were found at these sites.
As in trachea, AQP3 is present in the basal cells of the nasopharyngeal
epithelium (Fig.
4A). In
nasal conchus, however, anti-AQP3 strongly labels the basolateral
membrane of surface epithelial cells (Fig.
5A').
Immunoelectron microscopy confirmed extensive anti-AQP3 labeling of
basolateral plasma membranes in conchal surface epithelial cells (Fig.
6). Most of the surface epithelial cells
are labeled with anti-AQP3 (Figs.
5A' and 6), but closer
inspection revealed a few adjacent cells not surrounded by an
anti-AQP3-labeled plasma membrane, appearing as occasional large cells
with two nuclei (Fig. 5A');
these may correspond to the AQP4-labeled cells (Fig.
5B). Immunoelectron microscopy
confirmed the absence of AQP3 immunolabeling in these conchal
epithelial cells (Fig. 6). As in trachea, AQP4 is present in
basolateral membranes of ciliated surface epithelial cells in
nasopharynx (Fig. 4B); goblet cells
were not immunolabeled. Cells at the base of the pseudostratified
columnar epithelium of nasopharynx are predominantly unlabeled by
anti-AQP4 (Fig. 4B). AQP1 was not
detected in the surface epithelium of nasopharynx or conchus, but AQP1 is abundant in the capillaries and venules beneath the nasopharyngeal epithelium (Fig. 4D), as well as in
the capillaries and venous sinuses of the nasal conchus (Fig.
5D).

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Fig. 4.
Immunocytochemical localization of aquaporins in rat
nasopharyngeal surface epithelium (A-D)
and subepithelial glands (E-G). Cryosections (0.9 µm) were labeled with affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibody. A: basal cells of pseudostratified ciliated nasopharyngeal
epithelium are heavily labeled with anti-AQP3 (arrowheads). Except for
occasional cells that most likely represent sectioning artifact,
basolateral and apical membrane of ciliated surface epithelium (arrows)
are unlabeled. ×1,000. B: lateral plasma membranes of
nasopharyngeal surface epithelial cells strongly express AQP4 (arrows);
basal cells (arrowheads) of pseudostratified epithelium are
predominantly unlabeled. ×1,000 C: AQP5 is not
expressed in nasopharyngeal epithelium. ×1,000 D: AQP1
is not expressed in nasopharyngeal surface epithelium, but strong
immunolabeling is seen in both apical and basolateral plasma membranes
of endothelial cells in capillaries and venules (arrows). Subepithelial
fibroblasts (arrowheads) also express AQP1. ×1,000. E:
glands beneath nasopharyngeal epithelium exhibit no labeling with
anti-AQP3. ×480. F: immunolabeling control for G using nonimmune IgG. ×480. G: distinct
labeling with anti-AQP5 is noted in apical plasma membrane of glandular
epithelial cells (arrows), with no labeling of basolateral domains.
×1,000. This distribution was confirmed on multiple other
sections (not shown).
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Fig. 5.
Immunocytochemical localization of aquaporins in rat conchus epithelium
and glands. Cryosections (0.9 µm) were immunolabeled with
affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibodies. A and
A': AQP3 is strongly expressed
in basal and lateral plasma membranes (arrows) of most conchus surface epithelial cells (A') and
glandular epithelial cells (A).
Apical plasma membranes of conchus surface epithelium are not
immunolabeled (horizontal arrowheads, A'), and a few cells did
not label with anti-AQP3 (vertical arrowheads). ×480.
B: AQP4 is expressed in basolateral
membrane of only occasional cells in conchus surface epithelium and in
glandular epithelial cells (arrows). ×480.
C: AQP5 is expressed in apical
membrane of select clusters of cells within conchus epithelium
(arrows). ×480. D: conchus
surface epithelium is not labeled with anti-AQP1; endothelial cells in
capillaries and venules (arrows) exhibit strong labeling. ×480.
E: AQP4 immunolabeling is present in
basolateral membrane (arrows) of clusters of epithelial glandular cells
within surface epithelium with no labeling of apical membrane.
×1,000. F: parallel section to
(E) reveals expression of AQP5 in apical plasma membrane (arrows) of
glandular cell clusters. ×1,000.
G: heavy AQP3 immunolabeling is
visible in basolateral membrane of conchal subepithelial glandular
cells (small arrows); most cells in acinus are labeled, but apical
membrane is unlabeled (large arrows). ×1,000.
H: AQP4 immunolabeling is visible in
basolateral (small arrows) but not apical (large arrow) plasma membrane
of some subepithelial glandular cells of conchus. Some cells are not
labeled (arrowheads). ×1,000.
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Fig. 6.
Immunoelectron microscopic localization of AQP3 in rat conchal
surface epithelium. Ultrathin Lowicryl sections were labeled with
affinity-purified anti-AQP3 and visualized with goat anti-rabbit IgG
conjugated to 10-nm colloidal gold particles. Plasma membranes of
surface epithelial cells of conchal epithelium exhibit extensive AQP3
immunolabeling (arrows). Few cells within epithelium (marked with
asterisks) display no labeling. Labeling of surface epithelial cells is
seen also in plasma membrane processes interdigitating with other
surface epithelial cells. ×75,000.
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Cellular and subcellular distribution of aquaporins in nasal and
salivary glands.
In nasal conchus, AQP3 is strongly expressed in the basolateral
membrane of intraepithelial (Fig.
5A) and subepithelial glandular cells (Fig. 5, A and
G). AQP4 is expressed on the
basolateral membrane of some epithelial cells in nasal conchus
(scattered intraepithelial glands, Fig. 5,
B and
E). AQP4 is present on a larger
number of the subepithelial glandular cells (Fig.
5H) but is not present in all cells
of subepithelial glands. Thus, as in trachea, AQP3 and AQP4 are
expressed in nonoverlapping sites in nasopharynx and conchus (Figs. 4
and 5). In contrast, submandibular salivary glands did not label with
anti-AQP3 or anti-AQP4 (not shown); thus a mechanism for basolateral
water transport remains to be identified in salivary glands.
AQP5 is localized to the apical membrane of subepithelial glandular
cells in nasopharynx (Fig. 4G) but
is absent from the surface epithelium (Fig.
4C); specificity was confirmed by
nonimmune control (Fig. 4F).
Moreover, AQP5 is present in the apical plasma membrane of
intraepithelial glands of nasal conchus (Fig. 5,
C and
F) but is absent from subepithelial
glands (Fig. 5C). AQP5 is also
abundantly expressed in salivary glands (Fig.
7), as previously noted (15, 28). In the
submandibular salivary gland, AQP5 is present in the apical plasma
membrane of the secretory gland cells (Fig. 7,
A and
C). Moreover, AQP5 is present in the
secretory canaliculi and intercalated duct cells forming only the very
initial portion of the duct system (Figs. 7,
C and
D).

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Fig. 7.
Immunocytochemical localization of AQP5 in rat submandibular salivary
gland epithelium. Cryosections (0.9 µm) were labeled with
affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibodies. A: AQP5 is
confined to apical plasma membrane domains of apical surface of
glandular epithelium (arrows). No labeling is present in basolateral
aspects of cells. B: no labeling is
seen (arrows) in control for which nonimmune IgG was used as primary
antibody. C and
D: in addition to immunolabeling of
apical plasma membrane of secretory glandular epithelial cells
(arrows), prominent labeling is also seen over apical plasma membrane
domains of cells forming secretory canaliculi and very initial part of secretory duct (arrowheads).
A-D:
×1,000.
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DISCUSSION |
Fluid homeostasis in the lung and upper airway is complex, since myriad
anatomic and physiological factors influence the disposition of water.
Characterization of the relevant molecules is essential to understand
the regulation of water transport in the respiratory tract. In this
study, we demonstrate distinct, nonoverlapping distributions of AQP1,
AQP3, AQP4, and AQP5 from the alveolus to salivary glands and
nasopharynx in rat (Table 1).
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Table 1.
Summary of aquaporin cellular distribution in respiratory tract,
nasopharynx, nasal conchus, and oropharynx of rat
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The alveolar membrane has long been considered a tight epithelium,
restricting the movement of water and solute (11). Cellularity of the
membrane is maintained by differentiation of type II pneumocytes into
type I cells (1). Although AQP5 is specifically expressed in type I
pneumocytes, its absence in type II cells suggests a differentiation-specific signal, consistent with recent description of
AQP5 expression in a type II cell culture model (33). Water permeability was shown to be greater than solute permeability in the
alveolar membrane, suggesting that water crosses the membrane by a
transcellular rather than paracellular path (10). Mercury-inhibitable movement of water from the vascular space to the airspace was observed
in sheep, and it was proposed that AQP1 in type I pneumocytes mediates
water movement into the alveolus (12). Our demonstration of abundant
AQP5 in type I cells implicates this molecule in transalveolar water
movement, whereas AQP1 is not abundant in alveolar capillary endothelium of rat (18). Alterations in AQP5 expression or function may
participate in the pathogenesis or resolution of alveolar edema;
likewise, AQP5 may mediate the rapid absorption of water noted to occur
in the lungs of freshwater drowning victims. Other questions about
alveolar fluid dynamics still remain. It is uncertain whether salt
transporters are expressed in type I pneumocytes (20), and it is
unknown whether AQP5 in apical membranes of type I cells is
complemented by an unidentified water channel in the basolateral
membrane (Fig.
8A).

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Fig. 8.
Schematic diagrams representing aquaporins in rat airway and glandular
tissues. A: alveolus contains AQP5
(red) in apical membranes of type I pneumocytes
(P1); AQP1 (green) is sparsely present in underlying capillary endothelium. Although a pathway is
defined linking airspace and vascular space, no known aquaporin has
been identified in basolateral membrane of type I pneumocytes. P2, type II pneumocytes.
B: pseudostratified epithelium of
trachea and nasopharynx contains two populations of cells, basal cells that contain AQP3 (yellow) and columnar cells that reach airway surface
and contain AQP4 (blue). Subepithelial capillaries and fibroblasts
contain abundant AQP1. No known aquaporin has been identified in apical
membrane of surface epithelium. C:
secretory glands contain AQP5 in apical membranes but not in
basolateral membranes of acinar cells. Salivary glands do not contain a
known aquaporin in basolateral domains (not represented), whereas
glandular cells of nasopharynx and conchus contain AQP3 in basolateral
membranes of some acinar cells and AQP4 in basolateral membranes of
other acinar cells. Secretory canaliculi and intercalated cells forming very initial part of secretory duct contain abundant AQP5 but not AQP3
or AQP4 (not represented).
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AQP3 and AQP4 have been identified in tracheal and bronchial epithelium
(13), but our studies establish that these proteins have distinct,
nonoverlapping distributions in the ciliated, pseudostratified columnar
epithelium of trachea (Fig. 8B).
AQP3 is found only in basal cells of tracheal epithelium; these cells
are thought to serve as stem cells for the tissue and to anchor the
epithelium to the basement membrane (16). AQP3 is not expressed in lung distal to the trachea. In contrast, AQP4 resides in the basolateral membranes of columnar cells reaching the airway surface in trachea and
bronchi. The functional significance of this compartmentalization remains to be elucidated. AQP5 was identified in trachea by
immunoblotting (19) but was not visualized by histochemical analysis of
tracheal epithelium. We believe that AQP5 is expressed in glandular
epithelium in trachea, as in nasopharynx, but the relative paucity of
subepithelial glands in rat trachea (16) makes histological assessment
problematic.
We also demonstrate that AQP3, AQP4, and AQP5 have abundant,
nonoverlapping expression in the ciliated, pseudostratified columnar epithelium and subepithelial glands of nasopharynx and nasal conchus. Curiously, the patterns of expression of AQP3 and AQP4 are not identical. Cultured nasal epithelial cells have been reported to
rapidly change volume when hypertonic solutions were applied to the
luminal membrane (32), suggesting the existence of water channels at
the apical surface. We did not identify any of the known water channels
in the apical membrane of nasopharyngeal or conchal surface epithelium,
although AQP5 is present in the apical membrane of submucosal glandular
epithelium (Fig. 8C). Lack of a
known water channel at the apical membrane of nasopharyngeal surface
epithelium indicates that either a still unknown aquaporin resides at
that location or another mechanism may be responsible for water
transport at that site (Fig. 8B).
AQP1 is expressed on the apical and basolateral membrane of vessels
throughout the respiratory system. We previously demonstrated abundant
AQP1 in the bronchial circulation of the rat (18), a distribution
consistent with expression in subepithelial vessels of the trachea
shown here. Additionally, we now find abundant AQP1 expression in
subepithelial capillaries and venous sinusoids in nasopharynx, vessels
that in large part determine the resistance to airflow through the nose
(8). Because nasal capillaries and sinusoids are believed to be
fenestrated, AQP1 might play a role in cell volume regulation in
addition to its role in transcellular water movement (Figs. 8,
A and
B). The presence of AQP3 and AQP4 in
the basolateral membranes of surface epithelium of airways from
trachea, nasopharynx, and conchus suggests that these cells may
experience rapid increases or decreases in cell volumes without large
transcellular water flow (31). Alternatively, the presence of
aquaporins in the basolateral plasma membranes of respiratory epithelial cells may give this cellular surface high water
permeability, thereby preventing extensive changes in cell volume
during periodic loss of water from the apical plasma membrane from
airway humidification during inspiration and expiration.
Although specific functions remain speculative, the potential
physiological and pathophysiological significance of aquaporins in the
respiratory tract is considerable. Adequate gas exchange necessitates
tight control of water in the distal lung and alveolar space. Altered
expression or function of AQP5 or AQP1 in distal lung may contribute to
generation or amelioration of pulmonary edema. Alterations of the
airway surface layer may affect mucociliary transport and may also play
a role in the pathogenesis of exercise- or cold-induced asthma (3). It
is tantalizing to postulate that secondary alterations in aquaporin
expression or function could contribute to the pathogenesis of cystic
fibrosis (4) or provide a therapeutic mode for altering the viscosity
of airway secretions. Abundant expression of aquaporins in the
nasopharynx strongly suggests their participation in normal
physiological processes, such as humidification of inspired air, but
also suggests that alterations in their function or expression will
contribute to the pathogenesis of nasal congestion and rhinorrhea.
Appropriate management of fluid in vascular, interstitial, and airspace
compartments is essential for normal function of the respiratory system
throughout its length. Each of the aquaporin water channel proteins
present in the lung has a unique distribution and ontogeny, which
suggests functional specialization. In parallel to the extensive
investigation of solute transport in the lung (5), investigation of
water channels in this system is needed to fully understand the
physiology and pathophysiology of the respiratory tract.
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ACKNOWLEDGEMENTS |
We thank Hanne Weiling, Helle Bergmann, and Barbara L. Smith for
expert technical assistance.
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FOOTNOTES |
Support for this study was provided by the Novo Nordic Foundation, the
Karen Elise Jensen Foundation, the Danish Medical Research Council, and
the Biomembrane Research Center at the University of Aarhus (to S. Nielsen), National Institutes of Health Grants HL-33991, HL-48268, and
EY-11239 (to P. Agre), and National Research Service Award
HL-09119-02 (to L. S. King).
Addresses for reprint requests: P. Agre, Dept. Biological Chemistry,
Johns Hopkins University School of Medicine, 725 N. Wolfe St.,
Baltimore, MD 21205-2185; S. Nielsen, Dept. Cell Biology, Inst.
Anatomy, University of Aarhus, DK 8000 Aarhus C, Denmark.
Received 29 April 1997; accepted in final form 16 July 1997.
 |
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