Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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Cystic fibrosis is
caused by mutations in the cystic fibrosis transmembrane conductance
regulator (CFTR) Cl
channel, which mediates transepithelial
Cl
transport in a variety
of epithelia, including airway, intestine, pancreas, and sweat duct. In
some but not all epithelial cells, cAMP stimulates
Cl
secretion in part by
increasing the number of CFTR
Cl
channels in the apical
plasma membrane. Because the mechanism whereby cAMP stimulates CFTR
Cl
secretion is cell-type
specific, our goal was to determine whether cAMP elevates CFTR-mediated
Cl
secretion across serous
airway epithelial cells by stimulating the insertion of CFTR
Cl
channels from an
intracellular pool into the apical plasma membrane. To this end we
studied Calu-3 cells, a human airway cell line with a serous cell
phenotype. Serous cells in human airways, such as Calu-3 cells, express
high levels of CFTR, secrete antibiotic-rich fluid, and play a critical
role in airway function. Moreover, dysregulation of CFTR-mediated
Cl
secretion in serous
cells is thought to contribute to the pathophysiology of cystic
fibrosis lung disease. We report that cAMP activation of CFTR-mediated
Cl
secretion across human
serous cells involves stimulation of CFTR channels present in the
apical plasma membrane and does not involve the recruitment of CFTR
from an intracellular pool to the apical plasma membrane.
cystic fibrosis; serous cell; submucosal gland; chloride transport; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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CYSTIC FIBROSIS (CF), one of the most common lethal
autosomal recessive genetic diseases in Caucasians, is caused by
mutations in the cystic fibrosis transmembrane conductance regulator
(CFTR) gene, which encodes a multifunctional, integral membrane protein (9, 32, 33, 41). CFTR is a cAMP-activated
Cl channel, which mediates
transepithelial Cl
transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct (13). CF usually presents as exocrine pancreatic insufficiency, an increase in sweat
Cl
concentration, male
infertility, and airway disease. Airway disease leads to progressive
lung dysfunction, which is the leading cause of morbidity and mortality
in CF patients. Mutations in CFTR lead to impaired mucociliary
clearance in the lung as a result of reduced cAMP-activated
Cl
secretion by airway
epithelial cells, hyperabsorption of sodium and water, and production
of abnormal mucus and dehydration of the airways. The lungs of CF
patients become infected with Pseudomonas aeruginosa, setting in motion a cycle of inflammation,
tissue damage, impaired lung function, and eventually death (5).
It is well accepted that stimulation of CFTR-mediated
Cl secretion by cAMP
involves protein kinase A (PKA)-mediated phosphorylation of CFTR (13).
In addition, in some but not all epithelial cells, cAMP also stimulates
CFTR-mediated Cl
secretion
by increasing the amount of CFTR in the plasma membrane. For example,
in colon (T84 cells), kidney [Madin-Darby canine kidney (MDCK) II
and A6 cells], and shark rectal gland, as well as bronchial
epithelial cells, cAMP increases the amount of CFTR in the apical
membrane (1, 11, 18, 27, 40). By contrast, in gall bladder and T84
cells1
cAMP does not increase the amount of CFTR in the plasma membrane (6,
29, 39).
Serous cells in human airway submucosal glands express high levels of
CFTR, secrete antibiotic-rich fluid, and play a critical role in airway
function (8). Dysregulation of CFTR-mediated Cl secretion in serous
cells is thought to contribute to the pathophysiology of CF lung
disease (10, 35). However, it is not known whether cAMP regulates the
amount of CFTR in the apical plasma membrane of serous cells. Because
this issue has not been examined in serous cells and because the
mechanism whereby cAMP increases CFTR-mediated Cl
secretion is cell-type
specific, the goal of the present study was to determine whether cAMP
elevates Cl
secretion
across serous cells by increasing the amount of CFTR in the apical
plasma membrane. To this end we studied Calu-3 cells, a human airway
cell line with a serous cell phenotype (10, 12, 23, 35, 36).
Cl
secretion across Calu-3
cells is mediated by CFTR (10, 23, 36). We report that cAMP simulation
of CFTR-mediated Cl
secretion across Calu-3 cells involves activation of CFTR channels present in the apical membrane and does not involve the recruitment and
trafficking of CFTR Cl
channels from an intracellular compartment to the apical plasma membrane.
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METHODS |
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Cell culture. Calu-3 cells were obtained at passage 17 (HTB-55, American Type Culture Collection, Rockville, MD), cultured in tissue culture flasks (Costar, Cambridge, MA) coated with Vitrogen plating medium (VPM) containing DMEM (JRH Biosciences, Lenexa, KS), human fibronectin (10 µg/ml; Collaborative Biomedical Products, Bedford, MA), 1% Vitrogen 100 (Collagen, Palo Alto, CA), and BSA (10 µg/ml; Sigma Chemical, St. Louis, MO), and placed in an incubator maintained at 37°C and gassed with 5% CO2-95% air. Every 48 h, 90% of the medium (MEM, GIBCO-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM L-glutamine (GIBCO-BRL), 1 mM pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin (Sigma Chemical) was replaced. At 90% confluence, cells were subcultured by incubation in Hanks' balanced salt solution containing 0.05% trypsin and 0.53 mM EDTA (GIBCO-BRL) and reseeded at a 1:7 dilution in VPM-coated cell culture flasks or plated at 1 × 106 cells/0.6 cm2 on VPM-coated Millicell PCF filters and grown in air-water interface culture (23, 35). The medium was completely changed every 24 h when cells were grown on Millicell filters. Cells between passages 25 and 34 were studied 10-15 days after seeding. At 24 h before Ussing chamber experiments, FBS was removed from the cell culture medium. In these growth conditions we observed a higher transepithelial resistance (Rt), and the cells appeared structurally more differentiated than was observed previously (35; see RESULTS). In addition, in these culture conditions we did not observe any oscillations in baseline short-circuit current (Isc), as reported previously (35).
Measurement of Isc.
Isc was measured
by placing monolayers grown on Millicell PCF filters into an
Ussing-type chamber (Jim's Instrument, Iowa City, IA) and voltage
clamping the transepithelial voltage
(Vt) across the
monolayer to 0 mV with a voltage clamp (model VCC-600, Physiological
Instruments, Poway, CA), as described previously (15, 16, 28). Bath
solutions were maintained at 37°C and stirred by bubbling with 5%
CO2-95% air
(CO2/HCO3-containing
solutions) or room air
(CO2/HCO3-free
solutions). Current output from the clamp was digitized by a TL-1 DMA
Interface analog-to-digital converter (Axon Instruments, Foster City,
CA). To measure
Rt, a 1.0-mV
pulse (2-s duration) was imposed across the epithelium every 30 s, and
the resulting current deflection was measured. Rt was calculated
by Ohm's law: Rt = Vt/
Isc,
where
Vt is
1.0 mV. Data collection and analysis were done with Axotape 2.0 software (Axon Instruments).
Immunofluorescence and confocal microscopy.
Cells grown on Millicell PCF filters were immunostained for CFTR as
previously described (6). Briefly, cells were fixed with 3%
paraformaldehyde in PBS (Sigma Chemical) for 15 min, embedded in
Tissue-Tek (Miles, Elkhart, IN), frozen in liquid propane that was
cooled by liquid nitrogen, and stored at 80°C until further use. Sections (7-10 µm thick) were cut in a cryostat and placed on chrome alum gelatin-coated glass slides. Nonspecific binding sites
were blocked with PBS containing 10% goat serum (PBS-GS, Dako,
Carpinteria, CA), and sections were incubated with a 1:200 dilution of
an anti-CFTR regulatory domain (IgG1) or anti-CFTR COOH-terminal
(IgG2a) monoclonal antibodies (Genzyme, Cambridge, MA) in PBS with
0.5% BSA for 1-2 h at room temperature. Sections were washed with
PBS-1% BSA and incubated with a 1:25 dilution of goat anti-mouse
F(ab')2 fragment IgG-FITC
(Dako) in PBS with 0.5% BSA for 1 h at room temperature. The
immunolocalization pattern of CFTR with use of either antibody was
identical. The regulatory domain antibody has been used by others to
demonstrate that cAMP induces the recruitment of CFTR from a
cytoplasmic pool to the apical plasma membrane (27, 40). Thus this
antibody is capable of detecting changes in the intracellular
localization of CFTR.
Electron microscopy. Cells grown on Millicell PCF filters were fixed in 3.0% paraformaldehyde and 1.0% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4 adjusted to 300 mosM with sucrose) and stored in the fixative at 4°C until embedding into epoxy resin (Fluka, Buchs, Switzerland), as described previously (20). Briefly, filters were excised with a scalpel from the culture inserts, postfixed for 15 min in 1% OsO4, dehydrated in a graded series of ethanols, washed in propylene oxide, and flat embedded into epoxy resin. Ultrathin sections were cut with an ultramicrotome (Reichert Jung, Vienna, Austria) and stained with lead citrate and uranyl acetate. Ultrathin sections were viewed and studied with a Philips CM 100 electron microscope.
Apical membrane biotinylation. Biotinylation of apical cell membrane glycoproteins was performed as described in detail previously (19). All steps were performed at 4°C with gentle agitation. Cells grown on Millicell PCF filters were placed on ice and washed three times with ice-cold PBS (with 1 mM CaCl2 and 0.5 mM MgCl2, PBS-Ca2+/Mg2+) to block vesicular trafficking. Sugar residues of apical plasma membrane glycoproteins were oxidized with sodium m-periodate (10 mM) in PBS-Ca2+/Mg2+ added to the apical cell surface for 30 min. After three washes with ice-cold PBS-Ca2+/Mg2+ and one wash with 0.1 M sodium acetate buffer (pH 5.5), oxidized apical glycoproteins were biotinylated with biotin-LC-hydrazide (2 mM; Pierce Chemical, Rockford, IL) in 0.1 M sodium acetate buffer (pH 5.5) added to the apical cell surface for 30 min. Subsequently, cells were washed three times with PBS-Ca2+/Mg2+ and fixed for detection of biotin by Texas red-conjugated streptavidin or processed for quantitation of apical cell surface CFTR. Control experiments were performed by omitting the biotinylation step.
After biotinylation, cells were lysed with 75 µl of lysis buffer containing 50 mM Tris · HCl (pH 8.0), 150 mM NaCl, 1% NP-40, and protease inhibitors (Complete Protease Inhibitor Cocktail, Boehringer Mannheim, Indianapolis, IN), scraped from the filters, and transferred to an Eppendorff tube. Insoluble material was spun down by centrifugation at 14,000 g for 4 min. Supernatants were transferred to a new Eppendorff tube and brought to a total volume of 900 µl with lysis buffer, and biotinylated proteins were precipitated by adding 100 µl of a 50% suspension of streptavidin-agarose beads (Pierce). After overnight incubation at 4°C and continuous rotation, beads were pelleted by centrifugation for 30 s at 14,000 g and washed four times with lysis buffer. Biotinylated proteins were eluted from the beads by boiling for 5 min in 50 µl of Laemmli sample buffer [0.24 M Tris · HCl (pH 8.9), 16% glycerol, 0.008% bromphenol blue, 5.6% SDS, and 80 mM dithiothreitol] including 3% EDTA to inhibit oxidation of dithiothreitol during sample boiling. Eluates were subjected to SDS-PAGE in precasted 4-15% polyacrylamide gels (Bio-Rad) and transferred to polyvinylidine difluoride Immobilon membranes (Millipore, Bedford, MA). Membranes were blocked overnight at 4°C with 5% nonfat dry milk in Tris-NaCl buffer (10 mM Tris · HCl and 150 mM NaCl, pH 7.4) containing 0.02% Tween 20. Thereafter, membranes were incubated in the same buffer with the CFTR COOH-terminal antibody, diluted 1:500. After repeated rinsing in Tris-NaCl buffer, blots were incubated with an anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham, Arlington Heights, IL) diluted 1:5,000 in 5% nonfat dry milk and Tris-NaCl buffer. All antibody incubations were for 1 h at room temperature. After repeated rinsing, antibody-labeled proteins were visualized by the enhanced chemiluminescence detection method (Amersham) using Hyperfilm ECL (Amersham). Blots were digitally scanned, and the intensity of the bands was densitometrically analyzed with NIH Image version 1.57 software.Statistical analysis. Differences between means were compared by unpaired Student's t-tests or ANOVA followed by Bonferroni's multiple comparisons test as appropriate. All analyses were performed with the InStat statistical software package (Graphpad, San Diego, CA). Values are means ± SE. P < 0.05 is considered significant.
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RESULTS |
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8-(4-Chlorophenylthio)-cAMP stimulates electrogenic
Cl secretion by Calu-3 cells.
In unstimulated monolayers of Calu-3 cells bathed in MEM (
FBS),
Vt was
4.9 ± 0.6 mV,
Rt was 341 ± 32
· cm2,
and Isc was
19.6 ± 0.7 µA/cm2
(n = 26). 8-(4-Chlorophenylthio)-cAMP
(CPT-cAMP, 100 µM) rapidly increased
Isc, which
reached a peak value of 59.0 ± 3.0 µA/cm2
(n = 26) in ~2 min and then
decreased to a steady-state value of 51.0 ± 2.7 µA/cm2
(n = 26). The
Na+ channel blocker amiloride (10 µM) added to the apical bath solution had no effect on basal
Vt,
Rt, or
Isc or on
CPT-cAMP-stimulated Isc
(n = 13). Thus the basal and
CPT-cAMP-stimulated
Isc was not referable to electrogenic Na+
transport. To determine whether the CPT-cAMP-stimulated
Isc was mediated
by CFTR Cl
channels, we
examined the effects of the
Cl
channel blockers
diphenylamine-2-carboxylic acid (DPC),
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and DIDS added to
the apical bath solution on the CPT-cAMP-stimulated
Isc. DPC and NPPB
reduced the CPT-cAMP-stimulated Isc to basal
levels; however, DIDS had no effect on
Isc (Table 1). Inhibition by DPC and NPPB but not by
DIDS is a characteristic of CFTR
Cl
channels (36).
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Stimulation of Cl secretion by
CPT-cAMP is mediated by activation of CFTR
Cl
channels in the apical plasma
membrane.
If CPT-cAMP stimulates Cl
secretion in part by recruiting CFTR
Cl
channels from
intracellular vesicles to the apical plasma membrane and/or by
inhibiting the endocytotic retrieval of CFTR from the apical membrane
into cytoplasmic vesicles, activation of CFTR-mediated Cl
secretion should be
accompanied by or preceded by an increase in the amount of CFTR in the
apical cell membrane. Figure 1
demonstrates that CPT-cAMP had no effect on the cellular localization
of CFTR. CFTR was located almost exclusively in the apical cell
membrane in control and CPT-cAMP-treated monolayers.
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Microtubules are not involved in acute upregulation of CFTR-mediated
Cl secretion.
The regulated trafficking of many proteins from an intracellular pool
to the plasma membrane after an appropriate stimulus depends on intact
microtubules (2, 3, 13, 40). Moreover, constitutive trafficking of
proteins to the apical membrane is also microtubule dependent (17). To
determine whether CPT-cAMP-activated Cl
secretion is dependent
on intact microtubules, we treated cells with nocodazole (33 µM for 3 h before addition of CPT-cAMP), a drug that blocks microtubule
polymerization, or with taxol (10 µM for 3 h before addition of
CPT-cAMP), a drug that stabilizes and perturbs microtubule function (3,
34). Nocodazole completely disrupted microtubules (Fig.
5) but had no effect on CPT-cAMP-stimulated Isc (Table
3). Taxol caused microtubules to bundle and
thicken, but it had no effect on CPT-cAMP-stimulated
Isc (Table 3,
Fig. 5). Moreover, neither drug had an effect on the cellular
localization of CFTR (Fig. 5). In cells treated with nocodazole or
taxol, CFTR was localized to the apical plasma membrane. Thus
disruption of microtubule function for 3 h had no effect on basal or
cAMP-activated Cl
secretion
or on the localization of CFTR in the cell. These observations are
consistent with the view that cAMP activation of
Cl
secretion in human
airway epithelial cells is mediated by CFTR Cl
channels in the apical
plasma membrane and not by recruitment of CFTR
Cl
channels from an
intracellular pool to the apical plasma membrane.
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DISCUSSION |
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The major new finding of this report is that cAMP-mediated activation
of CFTR-mediated Cl
secretion across human airway serous epithelial cells (Calu-3) involves
activation of CFTR channels present in the apical membrane and does not
involve the recruitment of CFTR from an intracellular compartment to
the apical plasma membrane. Neither cAMP nor the PKA inhibitor H-89
altered the amount of CFTR in the apical plasma membrane, yet these
compounds dramatically influenced CFTR-mediated Cl
secretion. The inability
of the microtubule-disrupting agents nocodazole and taxol to alter the
Cl
secretory response to
cAMP or the cellular localization of CFTR is also consistent with our
conclusion that cAMP activation of CFTR-mediated
Cl
secretion across Calu-3
cells involves stimulation of CFTR channels present in the apical
membrane.
Taken together with previous studies, our data are consistent with the
conclusion that cAMP activation of CFTR-mediated
Cl secretion is cell-type
specific. In all cells examined, stimulation of CFTR by cAMP involves
PKA-mediated phosphorylation of CFTR. However, in kidney (MDCK II and
A6 cells) and shark rectal gland, as well as nasal and bronchial
epithelial cells, cAMP also enhances Cl
secretion by increasing
the amount of CFTR in the apical plasma membrane (1, 11, 18, 27, 37,
40). By contrast, in gall bladder and colon (T84 cells), cAMP does not
increase the amount of CFTR in the plasma membrane (6, 29, 39). It is well known that protein trafficking is cell-type specific, requiring numerous regulatory mechanisms and proteins that are expressed in some
but not all cells (7, 26). For example, the human LDL receptor, when
expressed in transgenic mice, is located in the apical membrane of
renal tubules and in the basolateral membrane of colonocytes and
enterocytes (3). The conclusion that the cellular mechanism whereby
cAMP activates CFTR-mediated
Cl
secretion is cell-type
specific has important implications for identifying therapeutic
strategies to treat CF. Thus it is imperative that studies be conducted
on epithelial cells affected by CF such as airway and pancreas, rather
than on heterologous expression systems. Moreover, because the delivery
of CFTR to the plasma membrane is dependent on the polarized state of
epithelial cells, it is prudent to study polarized cells. For example,
in unpolarized HT-29 intestinal cells, CFTR is localized in an
intracellular compartment, whereas in polarized HT-29 cells, CFTR is
sorted to the apical plasma membrane (24, 25).
Our studies with nocodazole and taxol also indicate that constitutive
trafficking of CFTR in Calu-3 cells is low compared with other
epithelial cells. For example, in T84 cells the half-life of CFTR in
the plasma membrane is 2 min (30), and cAMP doubles the amount of CFTR
in the apical membrane in 2 min (40). By contrast, in the present study
on Calu-3 cells, impairment of microtubule function for 3 h by
nocodazole or taxol had no effect on the amount of CFTR in the apical
plasma membrane. Because disruption of microtubules blocks the
intracellular flow of vesicles to the apical membrane of epithelial
cells but has no effect on the recycling of early endosomes (14), our
results with nocodazole and taxol suggest that CFTR has a very long
half-life in the plasma membrane of Calu-3 cells (3 h). This
conclusion is supported by preliminary studies with brefeldin A, a
fungal metabolite that inhibits the flow of vesicles from the
endoplasmic reticulum to the Golgi apparatus and, thereby, blocks the
delivery of newly synthesized proteins to the plasma membrane (34). We
found that brefeldin A had no effect on cAMP-activated
Cl
secretion across Calu-3
cells.
Why does cAMP regulate Cl
secretion in serous airway epithelial cells by activating channels that
are present in the membrane? Calu-3 and serous cells have high basal
rates of Cl
and antibiotic
secretion (23, 35). Thus relatively high and constant levels of CFTR in
the membrane are required to mediate basal and cAMP-activated
Cl
secretion.
In conclusion, we report that stimulation of
Cl secretion by human
serous airway epithelial cells (Calu-3) involves activation of CFTR
channels present in the apical plasma membrane and does not involve the
recruitment of CFTR from an intracellular compartment to the apical
plasma membrane. The observation that the cellular mechanism whereby
cAMP activates CFTR-mediated
Cl
secretion is cell-type
specific has important implications for studies directed at identifying
therapeutic strategies for CF.
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ACKNOWLEDGEMENTS |
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We are grateful to Alice Givan and Ken Orndorff for assistance with confocal microscopy and to Kerry O'Brien, Melissa Levak, and Lea Klausli for valuable technical assistance.
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FOOTNOTES |
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These studies were supported by National Institutes of Health Grant DK/HL-45881 and the Cystic Fibrosis Foundation. 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. 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).
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.
1 There is a discrepancy in the literature concerning the effect of cAMP on regulating the amount of CFTR in the apical plasma membrane in T84 cells (6, 29, 40). Although the reason is not clear, two points are relevant: 1) there are methodological differences among studies, and 2) immunolocalization of CFTR is technically challenging due in part to the low expression of CFTR in most epithelial cells (4, 42).
Address for reprint requests: B. A. Stanton, Dept. of Physiology, 615 Remsen Bldg., Dartmouth Medical School, Hanover, NH 03755.
Received 7 April 1998; accepted in final form 21 May 1998.
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