1 Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755; and 2 Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, and Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Serous cells secrete
Cl and HCO3
and play an important
role in airway function. Recent studies suggest that a
Cl
/HCO3
anion exchanger (AE) may
contribute to Cl
secretion by airway epithelial cells.
However, the molecular identity, the cellular location, and the
contribution of AEs to Cl
secretion in serous epithelial
cells in tracheal submucosal glands are unknown. The goal of the
present study was to determine the molecular identity, the cellular
location, and the role of AEs in the function of serous epithelial
cells. To this end, Calu-3 cells, a human airway cell line with a
serous-cell phenotype, were studied by RT-PCR, immunoblot, and
electrophysiological analysis to examine the role of AEs in
Cl
secretion. In addition, the subcellular location of AE
proteins was examined by immunofluorescence microscopy. Calu-3 cells
expressed mRNA and protein for AE2 as determined by RT-PCR and Western
blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat
tracheal serous cells in situ. In
Cl
/HCO3
/Na+-containing
media, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate
(CPT-cAMP)-stimulated short-circuit anion current (Isc) was reduced by basolateral but not by
apical application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
(50 µM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 µM)], inhibitors of AEs. In the absence of Na+ in the
bath solutions, to eliminate the contributions of the Na+/HCO3
and
Na+/K+/2Cl
cotransporters to
Isc, CPT-cAMP stimulated a small DNDS-sensitive Isc. Taken together with previous studies, these
observations suggest that a small component of cAMP-stimulated
Isc across serous cells may be referable to
Cl
secretion and that uptake of Cl
across
the basolateral membrane may be mediated by AE2.
cystic fibrosis; anion exchanger 2; serous cell; submucosal gland; chloride transport; sodium bicarbonate cotransport
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INTRODUCTION |
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AIRWAY EPITHELIAL CELLS
REGULATE the volume and composition of airway surface fluid by
mediating salt and water absorption and secretion (16, 32,
33). Pulmonary mucocillary clearance is dependent on the
thickness of the airway surface fluid and on the ability of cilia to
propel mucus out of the lungs. Adequate mucocillary clearance is
essential for the removal of inhaled particulate matter from the
airways and is vital for the prevention of airway tract infection and
obstruction. Submucosal glands play a major role in the secretion of
airway surface fluid (9, 21, 44). In particular, serous
cells in human airway submucosal glands express high levels of cystic
fibrosis transmembrane conductance regulator (CFTR), secrete
Cl and HCO3
, and fluid rich in
antibiotic defensin and maganin peptides (8, 9, 15, 44).
Moreover, dysregulation of CFTR-mediated Cl
secretion in
serous cells is thought to contribute to the pathophysiology of cystic
fibrosis lung disease (9, 45). However, the cellular mechanisms of fluid and electrolyte transport by human serous cells are
incompletely understood. Recent studies on Calu-3 cells, a human airway
cell line with a serous-cell phenotype, demonstrated that serous cells
secrete Cl
and HCO3
by electrogenic
processes (12, 17, 20, 24, 30, 36, 37).
HCO3
secretion is a two-step process: uptake across
the basolateral membrane is mediated by a
Na+/HCO3
cotransporter (NBC), and exit
from the cell is mediated by CFTR channels that are permeable to
Cl
and HCO3
(12, 20, 24).
Cl
secretion is also a two-step process: uptake across
the basolateral membrane is mediated by a
Na+/K+/2Cl
cotransporter (NKCC1),
and exit from the cell is mediated by CFTR Cl
channels
(12, 17, 20, 26, 27, 30, 37).
In many epithelia that secrete Cl, the uptake of
Cl
across the basolateral membrane is also mediated in
part by Cl
/HCO3
exchange (1, 2,
13, 43). The anion exchanger (AE, i.e., Cl
/HCO3
exchanger) gene family includes
three structurally and functionally related anion exchangers: AE1, AE2,
and AE3 (1, 2). AE1 is expressed at highest levels in red
blood cells and renal type A intercalated cells, AE2 is expressed
broadly in epithelia, and AE3 is expressed in brain and heart, as well
as in other excitable and some epithelial tissues. Recently, AE2 and
bAE3 (brain AE3) mRNA were detected in human airways; however, the cell
type and the subcellular location of AE2 and bAE3 in the airway is
unknown (13). Accordingly, the aim of the present study
was to test the hypothesis that AEs are expressed in submucosal glands,
particularly in serous cells, and that AEs contribute to
transepithelial Cl
secretion. To this end, we studied
Calu-3 cells, a human airway cell line with a serous-cell phenotype
(17, 20, 27, 30, 36, 37), and characterized the possible
role of AEs in Cl
secretion by RT-PCR, Western blot
analysis, and electrophysiological analysis. In addition, we examined
the cellular location of AE proteins in Calu-3 and rat serous cells by
immunofluoresence microscopy. We report that a small component of
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate
(CPT-cAMP)-stimulated short-circuit anion current
(Isc) across Calu-3 cells may be referable to
Cl
secretion and that Cl
uptake across the
basolateral membrane may be mediated in part by AE2.
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METHODS |
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Cell culture. Calu-3 cells were obtained at pass 17 from the American Type Culture Collection (HTB-55; Rockville, MD) and grown in air-interface culture (i.e., cell culture media added only to the basolateral side of cells grown on Millicell PCF filters) as described in detail previously (26, 27).
Measurement of Isc.
Isc was measured by placing monolayers grown on
Millicell polycarbonate (PCF) filters for 14-28 days in culture
into an Ussing-type chamber (Jim's Instrument, Iowa City, IA) as
described previously (26, 27). Bath solutions were
maintained at 37°C and were stirred by bubbling with 5%
CO2 air (CO2/HCO3-containing
solutions) or room air (CO2/HCO3
-free solutions).
Western blot analysis. Cell monolayers grown on Millicell filters and/or tissue culture flasks were solubilized in lysis buffer [50 mM Tris · HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and containing Complete Protease Inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN)] for 60 min at 4°C and spun at 14,000 g for 4 min to pellet insoluble material. Supernatants were separated on 4-20% Tris · HCl gradient gels (Bio-Rad) and transferred to polyvinylidene difluoride Immobilon membranes (Millipore, Bedford, MA). Membranes were blocked overnight at 4°C in 5% nonfat dry milk in Tris-buffered saline/0.02% Tween 20 and incubated with an affinity-purified polyclonal antibody (SA6) against the COOH-terminal 12 amino acids (1,224-1,237) of mouse AE2 (165 kDa). This antibody cross-reacts with another member of the AE anion exchanger family, AE1 (100-115 kDa), but does not recognize AE3 (at a 1:10,000 dilution) (3, 5, 6). After incubation with the primary antibody, blots were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Amersham, Arlington Heights, IL). Blots were developed by enhanced chemiluminescence using Hyperfilm ECL (Amersham). Other primary antibodies used to detect AEs included affinity-purified polyclonal antibodies against the COOH terminus of AE3 (SA8) and the cytoplasmic terminus of cardiac AE3 (cAE3; antibody no. SA17) (7).
Laser scanning confocal fluorescence microscopy.
Calu-3 cells were grown on Millicell PCF filters and were fixed and
processed for laser scanning confocal fluorescence microscopy as
previously described (26, 27). Briefly, cells were fixed with 3% paraformaldehyde for 15 min at room temperature and
subsequently embedded into cryoembedding compound (Microm, Walldorf,
Germany). Rat tracheas were harvested from four adult male Wistar rats
(BRL, Füllinsdorf, Switzerland) that weighed ~180 g. The rats
were anaesthetized with 100 mg/kg body wt of thiopental (Pentothal; Abbott, Cham, Switzerland), and the tracheas were fixed by
intravascular perfusion through the abdominal aorta. The
fixative solution consisted of 3% paraformaldehyde and 0.05% picric
acid in a 6:4 mixture of 0.1 M cacodylate buffer (pH 7.4, adjusted to
300 mosmol/kgH2O with sucrose) and 10% hydroxyethyl starch
in saline (HAES steril; Fresenius, Stans, Switzerland). After 5 min of
fixation, the fixative was washed out for 5 min with the
cacodylate-buffered solution. Tracheas were removed, cut into
~2-mm-thick slices horizontal to the long axis, and embedded into
cryoembedding compound. Embedded Calu-3 cells and tracheas were frozen
in liquid propane and stored at 80°C. Six-micrometer-thick sections
of frozen Calu-3 cells and rat tracheas were cut in a cryostat and
placed on chrome alum gelatin-coated glass slides. After SDS-antigen
retrieval [i.e., preincubation of the sections with 1% SDS for 10 min
(11)], nonspecific binding sites were blocked with 10%
normal goat serum (DAKO, Glostrup, Denmark) in PBS. Subsequently,
sections were incubated with 1:500-1:1,000 dilutions of polyclonal
AE antibodies (described above) in PBS with 0.5% BSA (PBS-BSA) for
1 h at room temperature. After repeated washings with PBS, binding
sites of the secondary antibody were revealed with a FITC-conjugated
swine-anti-rabbit IgG (DAKO) diluted 1:40 in PBS-BSA. To identify cell
nuclei, nucleic acids were stained with propidium iodide (2.5 µg/ml).
Sections were mounted in DAKO-Glycergel (DAKO) that contained 2.5%
1,4-diazabicyclo[2.2.2]octane to retard fading. Fluorescent images
were acquired using a Zeiss Axioskop microscope (Thornwood, NY)
equipped with a laser scanning confocal unit (model MRC-1024; Bio-Rad
Labs, Hercules, CA), a 15-mW krypton-argon laser, and a ×63
PlanApochromat/1.4 numerical aperture (NA) or ×40 PlanNeofluor/1.3 NA
oil-immersion objective. FITC fluorescence was excited using
the 488-nm laser line and collected using a standard FITC filter set
(530 ± 30 nm). Propidium iodide fluorescence was excited using
the 568-nm laser line and collected using a standard Texas Red filter
set (605 ± 32 nm). Acquired images were imported into Adobe
Photoshop version 3.0 for image processing and printing.
RT-PCR. mRNA was prepared from cells grown to confluency on Millicell PCF filters using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). Reverse transcription was performed with the First Strand DNA synthesis kit from Ambion. Each synthesis was loaded with 40 ng mRNA (estimated to correspond to 1 µg total RNA), and one-twentieth aliquots of the RT reaction were used in PCR reactions of 50 µl final volume. PCR was performed by the hot start procedure using Taq DNA polymerase (Promega) in the supplier's recommended buffer.
PCR mixes lacking only primers were preheated at 82°C for 1 min, and then gene-specific primers (Table 1) were injected into the mix through oil. The complete reaction mixes were denatured for 3 min at 95°C and then cycled through these conditions: denaturation for 45 s at 94°C, annealing for 2 min at 60°C, and elongation for 2 min at 72°C. Final extension of 10 min at 72°C was terminated by rapid cooling to 4°C after 35 cycles. PCR products were separated and analyzed in 1% agarose gels and sequenced to confirm their identities as specific AE cDNA fragments (ABI 373; Applied Biosystems, Foster City, CA).
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Statistical analysis. Differences between means were compared by either unpaired Student's t-tests or by analysis of variance followed by Bonferroni multiple-comparisons test as appropriate. All analyses were performed with the InStat statistical software package (Graphpad, San Diego, CA). Data are expressed as the means ± SE. P < 0.05 was considered significant.
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RESULTS |
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Molecular identification of AE isoforms in Calu-3 cells.
By RT-PCR, we determined that Calu-3 cells express mRNA for AE2 and low
levels of mRNA for bAE3. AE1 and cAE3 transcripts could not be detected
in Calu-3 cells (Fig. 1).
Negative controls for RT-PCR included omission of cDNA from the
reaction and yielded no product (data not shown). Positive controls for
AE1 and cAE3, including rat kidney and reticulocyte (AE1) and heart and
placenta (cAE3), were positive (Fig. 1). Thus Calu-3 cells express mRNA for AE2 and bAE3.
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Western blot analysis of AE isoforms in Calu-3 cells.
Three independent preparations of Calu-3 cells were assessed by
immunoblot for the presence of AE1, AE2, and AE3 polypeptides. As shown
in Fig. 2, a 165-kDa polypeptide was
detected by an antibody to the COOH terminus of AE2 (SA6). The 165-kDa
protein corresponding to AE2 was not detected when antibody incubations
were conducted in the presence of the immunizing peptide antigen. As
expected, on the basis of our RT-PCR studies, a polypeptide of the
appropriate molecular mass (100-115 kDa) could not be detected
with a mouse monoclonal antibody to rat AE1 (data not shown). Although
mRNA for bAE3 was identified, the expression of AE3 polypeptide could not be detected by immunoblot analysis using two different polyclonal antibodies (data not shown). Thus either Calu-3 cells do not express AE3 polypeptide, or the level of AE3 expression was below the sensitivity of our immunoblot analysis. Taken together, our RT-PCR and
Western blot studies demonstrate that Calu-3 cells express AE2 mRNA and
AE2 polypeptide.
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Immunolocalization of AE isoforms in Calu-3 and rat tracheal
epithelia.
Immunocytochemical localization of AE isoforms in Calu-3 cells and rat
trachea was performed using the same anti-AE antibodies that were used
for Western blot analysis. All Calu-3 cells and serous acinar cells of
rat submucosal glands examined had detectable AE2 polypeptide that was
localized to the basolateral plasma membrane (Fig.
3, E and F).
Furthermore, AE2 was seen in the basolateral membrane of some ciliated
surface epithelial cells but not in the epithelial cells lining the
ducts of submucosal glands (Fig. 3, A-D). AE2 was not
detected when antibody incubations were conducted in the presence of
the immunizing peptide. Consistent with our RT-PCR and Western blot
studies, AE1 polypeptide was not detected in Calu-3 cells or rat
submucosal glands using a monoclonal antibody specific for AE1
(14). However, as a positive control, the AE1 antibody did
stain intercalated cells in rat kidney as demonstrated previously (Ref.
3; results not shown). Thus Calu-3 and rat submucosal gland cells
express AE2 polypeptide in the basolateral but not the apical plasma
membrane.
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Electrophysiological studies: an AE in the apical membrane does not
contribute to Isc in Calu-3 cells.
To determine if AEs contribute to anion secretion by Calu-3 cells,
monolayers were mounted in an Ussing chamber, and the
CPT-cAMP-stimulated Isc was measured. In Calu-3
cells, Isc is referable to electrogenic Cl and/or HCO3
secretion (12, 17,
20, 26, 27, 30, 36, 37). Our immunolocalization studies suggest
that AE2 is expressed in the basolateral but not the apical membrane;
however, it is possible that an AE is present in the apical membrane,
but it cannot be detected by immunofluorescence microscopy. For
example, a functionally defined AE has been detected in the apical
membrane of
-intercalated cells in the renal cortical collecting
duct, but this AE cannot be detected with antibodies to AE1 or AE2
(3, 6, 40). Thus to explore the possibility that
functional AEs are present in the apical membrane, we determined
whether the anion exchange inhibitors
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), which inhibit Cl
/HCO3
exchange (1), and
dibenzamidostilbene disulfonic acid (DBDS), which inhibits
Cl
/OH
exchange (41), reduced
Isc. Although AEs are electroneutral, inhibition
of AE activity would be expected to alter Isc
indirectly by changing the intracellular activities of Cl
and HCO3
, and thus the electrochemical driving force
for Cl
and HCO3
exit across the apical
plasma membrane. Neither DIDS, DNDS, nor DBDS altered
Isc when added to the apical bath solution
(Table 2). Thus we could not
detect stilbene-sensitive AEs in the apical membrane of Calu-3 cells by
indirect immunofluoresence microscopy or by electrophysiological
techniques.
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Electrophysiological studies: an AE in the basolateral membrane
contributes to Isc in Calu-3 cells.
Ussing chamber studies were conducted to provide functional evidence
for an AE in the basolateral membrane of Calu-3 cells. We examined the
ability of the AE inhibitors DIDS and DNDS to inhibit
Isc, which is defined as an anion current
attributed to Cl and/or HCO3
secretion.
CPT-cAMP-increased Isc from 24.5 ± 3.0 µA/cm2 to 69.0 ± 4.2 µA/cm2 (Figs.
4 and
5A). DIDS (50 µM) and DNDS
(500 µM), when added separately to the basolateral bath solution,
reduced CPT-cAMP-stimulated Isc (Figs. 4 and
5A). DIDS (50 µM) completely inhibited the
CPT-cAMP-stimulated Isc (Fig. 4). DNDS (500 µM) also inhibited CPT-cAMP-stimulated Isc
but, unlike DIDS, did not reduce Isc to basal
levels (Figs. 4 and 5A). DIDS inhibits AE and the
Na+/HCO3
cotransporter (i.e., NBC). The
EC50 for DIDS inhibition of AE2 is 3-15 µM
(14, 18), whereas DIDS inhibition of NBC requires significantly higher concentrations (i.e., 500 µM) (10,
35). Accordingly, the observation that 50 µM DIDS completely
inhibited CPT-cAMP-stimulated Isc is consistent
with the view that an AE in the basolateral membrane contributes to
CPT-cAMP-stimulated Isc. However, we cannot
formally exclude the possibility that DIDS also reduced
Isc, in part, by inhibiting NBC in the
basolateral membrane. Thus additional studies were conducted to
determine whether a Na+-independent AE (i.e.,
Cl
/HCO3
exchanger) is present in the
basolateral membrane of Calu-3 cells (12).
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DISCUSSION |
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The major new findings in this report are that Calu-3 and serous cells in rat submucosal glands express AE2 in the basolateral membrane and that AE2 may contribute to electrogenic anion secretion across Calu-3 cells.
AE2 is expressed in the basolateral membrane of Calu-3 and serous cells in rat submucosal glands. Five lines of evidence in this report support the view that AE2 is expressed in Calu-3 cells: 1) abundant AE2 mRNA was detected by RT-PCR; 2) AE2 polypeptide was detected by Western blot analysis; 3) AE2 was localized to the basolateral membrane by indirect immunofluoresence; 4) in the absence of Na+, to eliminate the contribution of the NBC and NKCC1 to Isc, CPT-cAMP stimulated DNDS-sensitive Isc, and 5) DIDS, at a concentration well above the EC50 for AE2 (i.e., 4 µM) (14, 18, 19) and AE3 (23), inhibited the CPT-cAMP-stimulated Isc. In addition, AE2 was identified in the basolateral membrane of rat serous cells in airway submucosal glands. Thus AE2 was identified in the basolateral membrane of rat serous cells in vivo and in Calu-3 cells, a human airway cell line with a serous-cell phenotype (12, 17, 20, 30, 36, 37).
Neither pharmacological nor indirect immunofluoresence techniques identified AE2 in the apical membrane of Calu-3 or rat serous cells, in agreement with a recent study (37). Moreover, we did not find any evidence for AE1 expression in Calu-3 or rat serous cells. cAE3 mRNA was not expressed in Calu-3 cells; however, low levels of bAE3 mRNA were detected by RT-PCR. These observations are in agreement with a recent study in which AE2 and bAE3 mRNA was detected in human airways; however, the cell type expressing these AEs and the subcellular location of AE polypeptide was not determined (13). Thus our results extend these observations by localizing AE2 to the basolateral membrane of serous cells in the submucosal gland. Although mRNA for bAE3 was identified in Calu-3 cells, the expression of AE3 polypeptide could not be detected by immunoblot analysis using two different polyclonal antibodies. Thus Calu-3 cells may not express AE3 polypeptide. Alternatively, the level of AE3 polypeptide expression in the Calu-3 cell line may be below the sensitivity of our immunoblot analysis. AE2 has been localized to the basolateral membrane, but not the apical membrane, of numerous polarized epithelial cells including epididymis, stomach, small and large intestines, salivary glands, cochlea, vestibular apparatus, and alveolar type II cells (2, 5, 6, 11, 31, 39, 40, 47). bAE3 has been identified in the basolateral membranes of renal tubular cells (7) and has been observed on both basolateral and apical membranes of ileal and jejunal enterocytes (Stuart-Tilley, Wilhelm, and Alper, unpublished observations). cAE3 is expressed in the apical and basolateral membranes of renal epithelial cells, gut, and choroid plexus epithelia (Stuart-Tilley, Wilhelm, and Alper, unpublished observations). Thus although there is precedent for AE expression in apical membranes, our studies did not support the presence of an AE in the apical membrane of Calu-3 cells or in rat serous cells in situ.cAMP-stimulated Isc in Calu-3 cells.
In a previous study on Calu-3 cells, it was deduced from ion
substitution studies that forskolin-stimulated (i.e., cAMP-stimulated) Isc was referable to electrogenic
HCO3 secretion driven by a DNDS-sensitive,
Na+- and HCO3
-dependent cotransporter
(i.e., NBC) located in the basolateral membrane (12).
Removal of HCO3
or Na+ from the bath
solutions dramatically reduced the forskolin-stimulated Isc. Data in the present study confirm the
conclusion that the majority of cAMP-stimulated
Isc is referable to NBC-driven
HCO3
secretion in Calu-3 cells (12). We
observed that removal of Na+ from the bath solutions
dramatically reduced the CPT-cAMP-stimulated increase in
Isc from 44.5 to 7.5 µA/cm2. Our
data are also consistent with the view that a small component of
CPT-cAMP-stimulated Isc (~15% or 7.5 µA/cm2) is referable to Cl
secretion. In
the absence of Na+ in the bath solutions, to eliminate the
contribution of NBC and NKCC1 to Isc, CPT-cAMP
elicited a small increase in the DNDS-sensitive Isc (Fig. 5B). This DNDS-sensitive
current was dependent on HCO3
. These observations are
most consistent with the view that a small component (~15%) of
CPT-cAMP-stimulated Isc is mediated by
Cl
secretion, at least in the absence of Na+.
Several other laboratories have demonstrated that cAMP stimulates Cl36 efflux from Calu-3 cells and that the cAMP-stimulated
increase in Isc is equivalent to the increase in
Cl36 flux from the serosal to the mucosal solution across
Calu-3 cells (22, 25, 36). Moreover, in a recent study,
removal of Cl
from the bath solutions reduced
forskolin-stimulated Isc across Calu-3 cells
(from 66 to 46 µA/cm2), an observation consistent with
the view that cAMP stimulates some Cl
secretion across
Calu-3 cells (12). On the other hand, some laboratories
report that cAMP does not stimulate Cl
secretion across
Calu-3 cells (12, 24). Thus the effects of cAMP on
Cl
secretion across Calu-3 cells are controversial.
Cell model of electrogenic anion secretion by serous cells.
Figure 6 is a cellular model illustrating
the cellular location of transport proteins identified in Calu-3 and
serous cells that are relevant to Cl and
HCO3
transport. The location of these transport
proteins can account for Cl
and HCO3
secretion across serous cells in vivo (8). CFTR
Cl
channels are located in the apical membrane, and
NKCC1, Na+-K+-ATPase, NBC, and K+
channels are expressed in the basolateral membrane (12, 17, 20,
26, 27, 30, 37). In the present study, we have demonstrated that
AE2 is also expressed in the basolateral membrane. According to this
model, cAMP-stimulated HCO3
secretion is a two-step
process: uptake across the basolateral membrane is mediated by NBC, and
CFTR channels in the apical membrane mediate HCO3
exit from the cell (12, 20, 24). Cl
secretion is also a two-step process: uptake across the basolateral membrane is mediated by NKCC1, and Cl
exit from the cell
is mediated by CFTR Cl
channels (12, 17, 20, 26,
27, 30, 37). In addition, the data in this manuscript are
consistent with the conclusion that a small fraction of the
cAMP-stimulated increase in Isc may be referable
to Cl
secretion. We propose that Cl
uptake
across the basolateral membrane is mediated by AE2, and Cl
exits the cell through CFTR Cl
channels
in the apical membrane. We cannot exclude the possibility that AE2 may
also contribute to the regulation of intracellular pH. The relative
importance of each transport pathway to electrogenic anion secretion is
uncertain and may be dependent on the agonist stimulating the
secretion. However, this model is consistent with observations in
native tissue that cAMP stimulates Cl
and
HCO3
secretion by submucosal glands (8,
38).
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Alice Givan and Ken Orndorff for assistance with confocal microscopy.
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FOOTNOTES |
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These studies were supported by the National Institutes of Health (NIH) Grant DK-45881 and the Cystic Fibrosis Foundation (to B. A. Stanton), by NIH Grants DK-43495 and DK-34854 (Harvard Digestive Diseases Center, to S. L. Alper), and by National Research Service Award HL-09853 (to B. E. Shmukler). Confocal microscopy was performed at Dartmouth Medical School in the Herbert C. Englert Cell Analysis Laboratory, which was established by a grant from the Fannie E. Rippel Foundation and is supported in part by the Core Grant of the Norris Cotton Cancer Center (CA-23108).
J. Loffing was supported by a fellowship from the Swiss National Science Foundation. B. D. Moyer was supported by a predoctoral fellowship from the Dolores Zohrab Liebmann Foundation.
Address for reprint requests and other correspondence: B. A. Stanton, Dept. of Physiology, 615 Remsen Bldg., Dartmouth Medical School, Hanover, NH 03755 (E-mail: Bruce.A.Stanton{at}Dartmouth.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 7 December 1999; accepted in final form 27 April 2000.
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