PBA increases CFTR expression but at high doses inhibits
Cl
secretion in Calu-3
airway epithelial cells
Johannes
Loffing,
Bryan D.
Moyer,
Donna
Reynolds, and
Bruce A.
Stanton
Department of Physiology, Dartmouth Medical School, Hanover, New
Hampshire 03755
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ABSTRACT |
Sodium
4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to
treat patients with urea cycle enzyme deficiencies and is being
evaluated in the management of sickle cell disease, thalassemia,
cancer, and cystic fibrosis (CF). Because relatively little is known
about the effects of PBA on the expression and function of the
wild-type CF transmembrane conductance regulator (wt CFTR), the goal of
this study was to examine the effects of PBA and related compounds on
wt CFTR-mediated Cl
secretion. To this end, we studied Calu-3 cells, a human airway cell
line that expresses endogenous wt CFTR and has a serous cell phenotype.
We report that chronic treatment of Calu-3 cells with a high
concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but
not of sodium acetate reduced basal and
8-(4-chlorophenylthio)-cAMP-stimulated Cl
secretion.
Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and
increased the intensity of CFTR staining in the apical plasma membrane.
PBA also increased protein expression of
Na+-K+-ATPase.
PBA reduced CFTR Cl
currents across the apical membrane but had no effect on
Na+-K+-ATPase
activity in the basolateral membrane. Thus a high concentration of PBA
(5 mM) reduces Cl
secretion
by inhibiting CFTR Cl
currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05-2 mM) had no effect on cAMP-stimulated Cl
secretion across Calu-3
cells. We conclude that PBA concentrations in the therapeutic range are
unlikely to have a negative effect on
Cl
secretion. However,
concentrations >5 mM might reduce transepithelial Cl
secretion by serous
cells in submucosal glands in individuals expressing wt CFTR.
cystic fibrosis; cystic fibrosis transmembrane conductance
regulator; submucosal gland; chloride transport; gene expression; sodium-potassium-2 chloride cotransporter; sodium 4-phenylbutyrate
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INTRODUCTION |
SODIUM 4-PHENYLBUTYRATE (PBA), a short-chain
fatty acid that functions as an ammonia scavenger, has recently been
approved by the Food and Drug Administration to treat patients with
urea cycle enzyme deficiencies (29). Because PBA increases fetal hemoglobin levels, it is also being evaluated in the management of
sickle cell disease and thalassemia (6). PBA also causes cellular
differentiation and is used in phase I clinical trials as a
tumor-differentiating agent (12, 28, 29). PBA and its analog sodium butyrate have numerous and diverse cellular effects. These include modulation of protein kinase, phosphatase, and
ecto-5'-nucleotidase activities (7, 28, 30); stimulation of
microtubule and microfilament formation (1); inhibition of colonic
Cl
secretion (8); induction
of apoptosis (12); and transcriptional activation of numerous genes,
including heat shock proteins (12) and the cystic fibrosis
transmembrane conductance regulator (CFTR) (32, 33).
PBA is under evaluation as a treatment for cystic fibrosis (CF), a
systemic disease caused by mutations in the CFTR gene encoding a
cAMP-activated Cl
channel
(25, 32, 33). Seventy percent of individuals with CF express
F508
CFTR, a mutation that prevents the export of CFTR from the
endoplasmic reticulum to the apical plasma membrane, resulting in the
inability of cAMP to stimulate
Cl
secretion across
epithelial cells in the respiratory tract and pancreas (5, 11, 37, 37).
Overexpression of
F508 CFTR, a reduction in temperature, and
chemical chaperones allow some
F508 CFTR export out of the
endoplasmic reticulum to the plasma membrane, where
F508 CFTR
functions as a cAMP-activated
Cl
channel (9, 22, 34). PBA
increases
F508 CFTR expression and restores cAMP-activated
Cl
secretion in CF nasal
airway epithelial and CF bronchial epithelial cells (32, 33). Thus PBA
could be a useful therapy for CF in patients expressing
F508 CFTR.
Because little is known about the effects of PBA on wild-type (wt) CFTR
expression and function in epithelial cells, the goal of this study was
to examine the effects of PBA on wt CFTR-mediated Cl
secretion in airway
epithelial cells. To this end, we studied Calu-3 cells, a human airway
cell line with a serous cell phenotype (13, 15, 26, 35, 36). Serous
cells in human airway submucosal glands secrete
Cl
via CFTR located in the
apical cell membrane and secrete antibiotic-rich fluid (10).
8-(4-Chlorophenylthio)-cAMP (CPT-cAMP)-stimulated Cl
secretion across Calu-3
cells is a two-step process: uptake across the basolateral membrane is
mediated primarily by a
Cl
/HCO
3
exchanger, although an
Na+-K+-2Cl
cotransporter could also nominally contribute to
Cl
uptake, and secretion
across the apical membrane is mediated by CFTR (13, 15, 20, 26, 35,
36). We report that chronic treatment of Calu-3 cells with a high
concentration of PBA (5 mM) reduced basal and CPT-cAMP-stimulated
Cl
secretion.
Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and
increased the intensity of CFTR staining in the apical plasma membrane.
PBA also increased protein expression of
Na+-K+-ATPase.
However, PBA reduced CFTR-mediated
Cl
currents across the
apical membrane and had no effect on
Na+-K+-ATPase
activity in the basolateral membrane. Thus high concentrations of PBA
(5 mM) inhibit Cl
secretion
across Calu-3 cells primarily by inhibiting CFTR
Cl
currents across the
apical membrane. In contrast, lower therapeutic concentrations of PBA
had no effect on cAMP-stimulated
Cl
secretion across Calu-3 cells.
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METHODS |
Cell culture.
Calu-3 cells were obtained at passage 17 from the American
Type Culture Collection (Manassas, VA) and 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). Cells were then placed in an
incubator maintained at 37°C and gassed with 5%
CO2-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), was replaced. At 90% confluence,
cells were subcultured by incubation in Hanks' balanced salt solution
containing trypsin (0.05%) and EDTA (0.53 mM; 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
polycarbonate filters and grown in air-liquid interface culture (21,
26, 35). The medium was completely changed every 24 h when cells were
grown on Millicell filters. Cells were studied 10-15 days after
being seeded. Twenty-four hours before Ussing chamber experiments, FBS
was removed from the cell culture medium. Vehicle, sodium butyrate
(Sigma), PBA (Aldrich, Milwaukee, WI), valproic acid (Aldrich), or
sodium acetate (Sigma) was added to the cell culture medium, as
described (4, 8, 24, 32), from a filtered sterilized stock solution
prepared immediately before use every 1, 2, 4, or 6 days (as indicated in RESULTS).
Measurement of short-circuit current.
Short-circuit current
(Isc) was
measured across monolayers of Calu-3 cells in the presence of amiloride
(10
5 M) in the apical
solution to block the potential contribution of
Na+ transport to
Isc (16, 17, 27).
In the presence of amiloride, CPT-cAMP-stimulated
Isc in Calu-3
cells is equivalent to electrogenic Cl
secretion (21). Bath
solutions were maintained at 37°C and were stirred by bubbling with
5% CO2-air. Current output from the voltage clamp was digitized by a TL-1 DMA interface
analog-to-digital converter (Axon Instruments, Foster City, CA). Data
collection and analysis were done with Axotape 2.0 software (Axon
Instruments). During experiments, cells were bathed in an FBS-free MEM
solution containing (in mM) 116 NaCl, 24 NaHCO3, 3 KCl, 2 MgCl2, 0.5 CaCl2, 3.6 sodium HEPES, 4.4 hydrogen HEPES (pH 7.4), and 10 glucose.
To examine the effect of butyrate on the activity of the
Na+-K+-ATPase,
we permeabilized the apical membrane with nystatin (200 µg/ml) and
measured Isc, which, under these conditions,
represents the activity of Na+-K+-ATPase as
described previously (31). To examine the effect of butyrate on apical
membrane Cl
currents, we permeabilized the
basolateral membrane with nystatin (200 µg/ml) and measured
Isc in the presence of a transepithelial Cl
gradient directed from the apical to the
basolateral solution (140 vs. 14 mM;
Cl
was replaced on a
equimolar basis with gluconate;
Ca2+ was increased from 0.5 to 2 mM to maintain similar Ca2+
activity in both solutions) as described previously (8, 14). When the
basolateral membrane is permeabilized in the presence of a
transepithelial Cl
gradient, the Isc
is equal to the Cl
current
flowing across the apical membrane (8, 14).
Immunofluorescence and confocal
microscopy.
Cells were fixed with 3% paraformaldehyde in PBS for 15-30 min at
room temperature, embedded into cryo-embedding compound (Microm,
Walldorf, Germany), frozen in liquid nitrogen-cooled liquid propane,
and stored at
80°C until further use. Sections 6 µm thick
were cut in a cryostat and placed on chrome alum-gelatin-coated glass
slides. For immunocytochemical detection of CFTR, the tyramide signal
amplification kit (TSA-Direct; NEN, Boston, MA) was employed. After
SDS-antigen retrieval [i.e., preincubation of the sections with
1% SDS for 5 min (3)], nonspecific binding sites were blocked
with the TSA blocking buffer for 30 min at room temperature. Subsequently, sections were incubated with a 1:200 dilution of an
anti-CFTR regulatory domain (IgG1) monoclonal antibody (Genzyme, Cambridge, MA) in TSA blocking buffer for 1 h at room temperature. Similar results were obtained with a 1:200 dilution of a CFTR carboxy-terminal (IgG2a) monoclonal antibody (Genzyme).
Sections were washed with PBS containing 0.05% Tween 20 (PBS-Tween)
and incubated with a 1:100 dilution of horseradish
peroxidase-conjugated sheep anti-mouse IgG (Amersham, Arlington
Heights, IL) in TSA blocking buffer. After repeated washings with
PBS-Tween, binding sites of the secondary antibody were revealed with
FITC-tyramide conjugates diluted 1:50 in TSA diluent.
Na+-K+-ATPase
was detected with an
Na+-K+-ATPase
1-subunit monoclonal antibody (diluted 1:1,000; Upstate Biotechnology, Lake Placid, NY) followed by a 1:25 dilution of a goat
anti-mouse F(ab')2 fragment IgG-FITC (DAKO). To
detect the Na+-K+-2Cl
cotransporter, sections were preincubated with 1% SDS for 10 min at
room temperature and then incubated with monoclonal antibody T4
(1:2,000), generously provided by Dr. B. Forbush (23), followed by a
1:25 dilution of a goat anti-mouse F(ab')2 fragment
IgG-FITC (DAKO). To identify cell nuclei, nucleic acids were stained
with propidium iodide (2.5 µg/ml). Sections were mounted in
DAKO-Glycergel (DAKO, Carpinteria, CA) containing 2.5%
1,4-diazabicyclo- [2.2.2]octane to retard fading. Acute
exposure to PBA (40 min) had no effect on the immunocytochemical
localization of CFTR,
Na+-K+-ATPase,
or the
Na+-K+-2Cl
cotransporter compared with untreated cells. Moreover, in cells treated
with PBA for 6 days, removal of PBA from the cell culture medium 2 h
before processing for immunocytochemistry also did not alter the
immunolocalization of the transporters compared with cells treated
continuously with PBA for 6 days.
Fluorescent images were acquired by using a Zeiss confocal
laser-scanning microscope (LSM 310; Carl Zeiss, Oberkochen, Germany) equipped with a ×63 PlanApochromat/1.4-NA oil-immersion
objective. FITC fluorescence was excited with the use of the 488-nm
argon laser line and collected by using a 515- to 570-nm band-pass
filter. Propidium iodide fluorescence was excited by using the 543-nm helium-neon laser line and collected with the use of a 575-nm long-pass
filter. All images were acquired by using the same confocal parameters
and were imported into Adobe Photoshop version 3.0 for processing and printing.
Western blot analysis.
Cell monolayers were solubilized in lysis buffer [50 mM
Tris · HCl, pH 8.0, 150 mM NaCl, and
1% NP-40, containing the complete protease inhibitor
cocktail (Boehringer Mannheim, Indianapolis, IN)] for 60 min at
4°C, scraped from filters, and spun at 14,000 g for 4 min to pellet insoluble
material. Supernatants were separated on 4-15%
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-0.1% Tween 20 and incubated with either the CFTR
carboxy-terminal monoclonal antibody (1:1,000), Na+-K+-ATPase
1-subunit monoclonal antibody (1:1,000), or
Na+-K+-2Cl
monoclonal antibody T4 (1:10,000), followed by anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000; Amersham). Blots
were developed by enhanced chemiluminescence (Amersham) with the use of
Hyperfilm ECL (Amersham). Densitometric analysis of band intensities
was performed with public domain National Institutes of Health Image
version 1.57 software.
Statistical analysis.
Differences between means were compared by either paired or unpaired
Student's t-tests or ANOVA, followed
by Bonferroni multiple comparisons test as appropriate. Analyses were
performed with the InStat statistical software package (Graphpad, San
Diego, CA). Data are expressed as means ± SE. P < 0.05 was considered significant.
 |
RESULTS |
PBA, sodium butyrate, and sodium valproate inhibit
Cl
secretion.
As we previously demonstrated (21), CPT-cAMP rapidly increased
Isc, which
reached a peak in 2 min and then declined to a steady-state value
significantly above basal
Isc (Fig.
1). As illustrated in Figs.
1-4, PBA and sodium butyrate decreased
basal and CPT-cAMP-stimulated
Isc after 1, 2, and 6 days of continuous treatment. In
contrast, acute exposure to PBA or sodium
butyrate (10 min before addition of CPT-cAMP) had no effect on basal or CPT-cAMP-stimulated
Isc (Fig.
5). We also examined the effect of sodium
valproate on Isc
to determine whether other fatty acids also inhibit
Isc. Sodium
valproate is an anticonvulsive that is also used as a mood stabilizer
in individuals with bipolar disorder (2). Sodium valproate at
clinically relevant concentrations (i.e., 100 µM to 1 mM for 2 days)
had no effect on basal or CPT-cAMP-stimulated Cl
secretion by Calu-3
cells (n = 6 monolayers/group). However, at 5 mM, sodium valproate (2 days) inhibited basal and CPT-cAMP-stimulated Isc (Fig.
6). At a comparable concentration (5 mM),
sodium valproate elicited a larger inhibition of basal and
CPT-cAMP-stimulated Isc
than PBA and sodium butyrate (compare Figs. 2-4 with Fig. 6). In
contrast, continuous exposure to sodium acetate (5 mM) for 2 days
had no effect on basal or CPT-cAMP-stimulated
Isc (Fig. 7).

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Fig. 1.
Representative short-circuit current
(Isc) records
illustrating effect of sodium 4-phenylbutyrate (PBA) and sodium
butyrate (Na-butyrate) on basal and 8-(4-chlorophenylthio)-cAMP
(CPT-cAMP)-stimulated
Isc. Three
Isc traces are
shown: control, PBA (5 mM, 6 days), and sodium butyrate (5 mM, 6 days).
CPT-cAMP (100 µM) was added to both apical and basolateral bathing
solutions at time indicated by arrow. Measurements of
Isc in steady
state were taken after oscillations in Isc were
minimal (<0.5 µA/cm2; ~12 min after addition of
CPT-cAMP). Mean transepithelial resistance
(RT) increased,
as expected when
Isc falls, with
butyrate treatment. However, the change did not achieve statistical
significance in all cases. For example,
RT was 272 ± 35 · cm2 in
control monolayers (n = 26) and 324 ± 32 · cm2 in
monolayers treated with sodium butyrate for 6 days
(n = 12, P = not significant).
RT increased
significantly from 272 ± 35 · cm2 in
control monolayers (n = 26) to 714 ± 82 · cm2 in
monolayers treated with PBA for 6 days
(n = 14, P < 0.01).
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Fig. 2.
Effect of sodium butyrate (NaB) and PBA on basal
Isc. For data
summarized in Figs. 2-4, no. of monolayers in each group was 26 for control, 7 for sodium butyrate 1 day (1 d), 6 for sodium butyrate 2 d, 12 for sodium butyrate 6 d, 6 for PBA 1 d, 6 for PBA 2 d, 7 for PBA
4 d, and 14 for PBA 6 d. In this and subsequent figures, basal
Isc is defined as
current measured 1 min before addition of CPT-cAMP to apical and
basolateral bath solutions.
Isc was identical
in all sets of control monolayers. Thus in Figs. 2-5, we pooled
control data to simplify presentation.
* P < 0.05 vs. control.
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Fig. 3.
Effect of sodium butyrate and PBA on CPT-cAMP-stimulated
Isc (peak value).
In this and subsequent figures, peak
Isc is defined as
current measured at peak increase after addition of CPT-cAMP (100 µM)
to apical and basolateral bath solutions.
* P < 0.05 vs. control.
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Fig. 4.
Effect of sodium butyrate and PBA on steady-state
Isc. In this and
subsequent figures, steady-state
Isc is defined as
current measured 10-12 min after decline of
Isc from peak
value after addition of CPT-cAMP (100 µM) to apical and basolateral
bath solutions. * P < 0.05 vs.
control.
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Fig. 5.
Effect of acute exposure to sodium butyrate and PBA on
Isc. Sodium
butyrate (5 mM) or PBA (5 mM) was added to apical and basolateral bath
solutions 10 min before basal
Isc was measured.
Hatched bars, control data (NaCl, 5 mM;
n = 10); open bars, PBA data
(n = 10); gray bars, sodium butyrate
data (n = 10). SS, steady state.
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Fig. 6.
Effect of sodium valproate on basal and CPT-cAMP-stimulated
Isc. Hatched
bars, control (vehicle, 0.1% ethanol, for 2 days;
n = 4); open bars, sodium valproate (5 mM, for 2 days; n = 4).
* P < 0.0001 vs. control.
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Fig. 7.
Effect of sodium acetate on basal and CPT-cAMP-stimulated
Isc. Hatched
bars, control (vehicle, distilled water;
n = 4); open bars, sodium acetate (5 mM, for 2 days; n = 4).
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Dose-response studies: PBA and
Cl
secretion.
Rubenstein and Zeitlin (33) report that a therapeutic effect of PBA was
observed in CF patients when plasma concentrations of PBA were between
0.05 and 2 mM. To determine whether therapeutic concentrations of PBA
inhibit Cl
secretion across
Calu-3 cells, we examined the effect of PBA at concentrations between
0.05 and 2 mM for 4 days. As shown in Fig.
8, concentrations of PBA between 0.05 and 2 mM had no effect on CPT-cAMP-stimulated
Cl
secretion. However, as
reported above, 5 mM PBA reduced CPT-cAMP-stimulated Cl
secretion (Fig. 8). Thus
therapeutic concentrations of PBA have no effect on CPT-cAMP-stimulated
Cl
secretion across Calu-3
cells.

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Fig. 8.
Dose-response studies: PBA and CPT-cAMP-stimulated
Cl currents. Monolayers
were treated with vehicle (distilled water, labeled control) or PBA
(0.05-5 mM) for 4 days. Hatched bars, peak
Isc after
CPT-cAMP (100 µM); gray bars, steady-state
Isc after
CPT-cAMP (100 µM); n = 7-13
monolayers/group. * P < 0.001 vs. control.
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Effect of PBA on CFTR,
Na+-K+-ATPase,
and
Na+-K+-2Cl
expression.
We examined the possibility that PBA (5 mM) might decrease
CFTR-mediated Cl
secretion
by altering the expression of key transport proteins involved in
transepithelial Cl
secretion in Calu-3 cells, including CFTR,
Na+-K+-ATPase,
and the
Na+-K+-2Cl
cotransporter (35). CFTR mediates
Cl
secretion across the
apical cell membrane, and Na+-K+-ATPase
maintains a low intracellular concentration of
Na+, which is important for
providing the driving force for
Cl
entry across the
basolateral membrane primarily by
Cl
/HCO
3
exchange. However, the
Na+-K+-2Cl
cotransporter also contributes to
Cl
uptake (35). Operation
of the Cl
/HCO
3
exchanger and the Na+-K+-2Cl
transporter maintains
Cl
above the apical
membrane electrochemical equilibrium potential, thereby establishing
the driving force for Cl
secretion across the apical membrane into the airway surface fluid
(35). Western blot analysis revealed that PBA and sodium butyrate
increased CFTR and
Na+-K+-ATPase
protein levels (Figs. 9 and
10). The increase in transport protein
expression was surprising, given the observed decreased in
Cl
secretion. In contrast,
PBA decreased
Na+-K+-2Cl
cotransporter expression (Figs. 9 and 10).


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Fig. 9.
Effect of PBA and sodium butyrate on cystic fibrosis transmembrane
conductance regulator (CFTR),
Na+-K+-ATPase,
and
Na+-K+-2Cl
cotransporter protein expression. Protein expression was determined by
Western blot analysis as described in
METHODS. Data are arbitrary
densitometry units where control values have been normalized to 1. A: CFTR after 6 days of treatment with
PBA (5 mM) or sodium butyrate (5 mM). No. of monolayers studied was 16 for control (CON), 12 for PBA, and 8 for sodium butyrate.
* P < 0.01 vs. control.
B:
Na+-K+-ATPase
after 6 days of treatment with PBA (5 mM) or sodium butyrate (5 mM).
No. of monolayers studied was 9 for control, 9 for PBA, and 5 for
sodium butyrate. * P < 0.01 vs. control. In time-course experiments, PBA and sodium butyrate
significantly increased CFTR and
Na+-K+-ATPase
expression after 1 day of treatment, and increase reached a maximum
value after 4 days of treatment (n = 3). C:
Na+-K+-2Cl
cotransport after 6 days of treatment with PBA (5 mM). No. of
monolayers studied was 3 for control and 3 for PBA.
* P < 0.01 vs. control.
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Fig. 10.
Representative Western blots demonstrating that PBA and sodium butyrate
increased CFTR and
Na+-K+-ATPase
protein expression and that PBA decreased
Na+-K+-2Cl
protein expression in Calu-3 cells. Equal amounts of protein were added
to each well of gel (40 µg). See methods and legend to Fig.
9 for details on experimental protocol.
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To provide additional support for the observation that sodium butyrate
and PBA altered the expression of CFTR,
Na+-K+-ATPase,
and the
Na+-K+-2Cl
cotransporter, we performed immunofluorescence microscopy studies. As
described previously (21), CFTR was found in the apical plasma membrane
of Calu-3 cells (Fig. 11,
A and
B). The vast majority of control
cells expressed undetectable or very low levels of CFTR (Fig.
11A). Treatment with PBA (5 mM for
6 days) increased the intensity of CFTR immunostaining in the apical
plasma membrane (Fig. 11, compare A
with B). PBA also increased the
intensity of Na+-K+-ATPase
staining, which was localized predominantly in the lateral membrane in
control and PBA-treated monolayers (Fig. 11,
C and D). Thus PBA increased the amount of
CFTR in the apical membrane and
Na+-K+-ATPase
in the basolateral membrane. In contrast, PBA decreased the intensity
of
Na+-K+-2Cl
staining in the basolateral membrane (Fig. 11, compare
E with F). These immunofluorescence
microscopy studies are consistent with our Western blot analyses,
demonstrating that PBA increased CFTR and
Na+-K+-ATPase
expression but decreased
Na+-K+-2Cl
cotransporter expression.

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Fig. 11.
Effects of PBA on expression and localization of CFTR,
Na+-K+-ATPase, and
Na+-K+-2Cl cotransporter.
A: immunolocalization of CFTR in control cells.
B: immunolocalization of CFTR in cells
treated with PBA (5 mM) for 6 days. A
and B: CFTR is red, and nuclei,
stained with propidium iodide, are purple. PBA increased intensity of
CFTR staining in apical plasma membrane and number of cells expressing
CFTR. C: immunolocalization of
Na+-K+-ATPase
in control cells. D:
immunolocalization of
Na+-K+-ATPase
in cells treated with PBA (5 mM) for 6 days.
C and
D:
Na+-K+-ATPase
is green, and nuclei are purple. PBA increased intensity of
Na+-K+-ATPase
staining in lateral membrane. E:
immunolocalization of
Na+-K+-2Cl
cotransporter in control cells. F:
immunolocalization of
Na+-K+-2Cl
cotransporter in cells treated with PBA (5 mM) for 6 days.
E and
F:
Na+-K+-2Cl
cotransporter is green, and nuclei, stained with propidium iodide, are
purple. PBA decreased intensity of
Na+-K+-2Cl
cotransporter staining in basolateral plasma membrane. For each
transport protein examined, images in control and PBA-treated cells
were obtained by using identical settings on confocal microscope and
were printed with the use of same settings. It is important to note
that Calu-3 cells maintained a polarized epithelium, as revealed in
these images, capable of secreting
Cl in a regulated manner
(i.e., CPT-cAMP) even when exposed to a relatively high concentration
of PBA (5 mM). Scale bar = 10 µm.
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Effect of PBA on
Na+-K+-ATPase
activity and Cl
- channel currents.
To examine the effect of PBA on the activity of
Na+-K+-ATPase, we permeabilized the apical
membrane with nystatin and measured Isc, which, under
these conditions, represents the activity of Na+-K+-ATPase
(31). PBA had no effect on
Na+-K+-ATPase
activity. Isc was 106.1 ± 3.5 µA/cm2 (n = 4) in control monolayers and 119.0 ± 6.9 µA/cm2 in monolayers exposed to PBA (5 mM for 2 days; n = 4, P = 0.20). Thus, although PBA
increased
Na+-K+-ATPase
protein expression, the drug had no effect on
Na+-K+-ATPase
activity. To examine the effect of PBA on apical membrane CFTR
Cl
currents, we
permeabilized the basolateral membrane with nystatin and measured
Isc in the presence of a transepithelial
Cl
concentration gradient directed from the apical
to the basolateral solution. PBA dramatically reduced the
Isc flowing
across apical CFTR Cl
channels from 63.7 ± 10.4 µA/cm2 (n = 6) in control monolayers to 19.1 ± 2.1 µA/cm2 in monolayers treated with PBA (5 mM PBA
for 2 days; n = 6, P < 0.01). Thus, although PBA
increased CFTR protein expression, the drug reduced the CFTR-mediated
Cl
currents. Taken
together, the Western blot, confocal microscopy, and Ussing chamber
studies suggest that PBA reduces CFTR-mediated Cl
secretion in part by
inhibiting the activity of CFTR
Cl
channels in the apical membrane.
 |
DISCUSSION |
The major new finding of this report is that chronic treatment with the
short-chain fatty acids PBA, sodium butyrate, and sodium valproate but
not sodium acetate at a concentration of 5 mM reduced basal and
CPT-cAMP-stimulated Cl
secretion across polarized human airway epithelial cells (Calu-3) expressing wt CFTR. Paradoxically, PBA and sodium butyrate increased the expression of CFTR and
Na+-K+-ATPase.
However, PBA reduced CFTR
Cl
currents across the
apical membrane. In contrast, lower therapeutic concentrations of PBA
(0.05-2 mM) had no effect on cAMP-stimulated Cl
secretion across Calu-3
cells. We conclude that PBA concentrations in the therapeutic range are
unlikely to have a negative effect on
Cl
secretion. However,
concentrations >5 mM might reduce transepithelial Cl
secretion by serous
cells in submucosal glands in individuals expressing wt CFTR.
Sodium butyrate also inhibits electrogenic
Cl
secretion across
intestinal cell lines. In T-84 cells, a human colonic epithelial cell
line, chronic sodium butyrate treatment (i.e., days) decreased cAMP-stimulated Cl
secretion (18, 24). The inhibition of
Cl
secretion by sodium
butyrate was mediated by a reduction of
Na+-K+-2Cl
cotransporter expression: 125I efflux through apical
membrane CFTR Cl
channels
was not affected by sodium butyrate (24). In contrast, chronic sodium
butyrate treatment had no effect on cAMP-activated Cl
efflux across HT-29
cells, a human colonic epithelial cell line (24). Acute exposure (i.e.,
minutes) to sodium butyrate inhibited cAMP-stimulated
Cl
secretion by rat colon
(8) and T-84 cells by reducing apical membrane
Cl
conductance (8). In
contrast, in the present study, acute treatment with sodium butyrate
had no effect on cAMP-stimulated Cl
secretion. Although the
reason(s) for these divergent observations is not clear, it is possible
that the effects of sodium butyrate might be tissue specific as well as
time and dose dependent.
Inhibition of CFTR-mediated
Cl
secretion across Calu-3
cells by PBA and sodium butyrate was not observed at clinically
relevant concentrations [i.e., 0.05-2 mM (29, 33)].
Thus it is unlikely that PBA given to individuals with urea cycle
enzyme deficiencies, sickle cell disease, thalassemia, or cancer would
have the untoward effect of inhibiting
Cl
secretion across
epithelial cells expressing wt CFTR. Sodium valproate at clinically
relevant concentrations (i.e., 100 µM to 1 mM for 2 days) also had no
effect on basal or CPT-cAMP-stimulated Cl
secretion by Calu-3
cells. Inhibition of Cl
secretion by sodium valproate was observed only at 5 mM. Thus it is
unlikely that valproic acid therapy would alter
Cl
secretion in vivo in
epithelial cells expressing wt CFTR.
In this report, we began to examine some of the possible mechanisms
whereby PBA (5 mM) inhibits CFTR-mediated
Cl
secretion across Calu-3
cells. We examined the possibility that PBA reduced
Cl
secretion by
downregulating the expression of transport proteins involved in
Cl
secretion across Calu-3
cells, including CFTR,
Na+-K+-ATPase,
and the
Na+-K+-2Cl
cotransporter (19, 24, 36). Paradoxically, we found that PBA increased
CFTR and
Na+-K+-ATPase
protein expression, effects that would be expected to stimulate, not
inhibit, Cl
secretion.
However, our Ussing chamber studies revealed that PBA reduced
CFTR-mediated Cl
currents
across the apical membrane but had no effect on the activity of
Na+-K+-ATPase. Thus the major mechanism whereby
PBA reduces Cl
secretion
involves inhibition of apical CFTR-mediated
Cl
currents, even though
there are more CFTR Cl
channels in the apical membrane. This later observation suggests that
PBA could have dual effects on CFTR:
1) to increase the number of
channels in the membrane and 2) to
reduce the current flow through CFTR
Cl
channels. The mechanism
of this later effect is unknown and requires additional studies.
It also must be considered that a reduction in
Na+-K+-2Cl
cotransporter and/or
Cl
/HCO
3
exchange expression in the basolateral membrane might also contribute
to the reduction in Cl
secretion observed in PBA-treated cells. Although we report that PBA
reduced
Na+-K+-2Cl
expression, this transporter contributes only nominally to
CPT-cAMP-stimulated Cl
secretion by Calu-3 cells (13, 15, 20, 26, 35, 36). Thus it is unlikely
that the reduction in
Na+-K+-2Cl
cotransporter protein expression plays a major role in the inhibition by PBA of Cl
secretion.
Additional studies, including extensive investigation to define more
completely the roles of the
Cl
/HCO
3
exchanger and the
Na+-K+-2Cl
cotransporter, are required to elucidate the mechanism whereby PBA
inhibits CFTR-mediated Cl
secretion.
In conclusion, the observation that PBA (5 mM) has a negative effect on
wt CFTR-mediated, cAMP-stimulated
Cl
secretion in Calu-3
cells, a model cell type for human serous cells in submucosal glands in
the trachea, does not appear to have implications for the clinical use
of this drug for patients with CF, urea cycle enzyme deficiencies,
sickle cell disease, thalassemia, and cancer as long as the therapeutic
concentrations are in the range of 0.05-2 mM. However, we conclude
that plasma PBA levels of 5 mM or above in individuals expressing wt
CFTR could have untoward effects on transepithelial
Cl
secretion by serous
cells in the submucosal glands.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Jack McBain for helpful suggestions; Dr. David McCoy
and Collin Shaw for performing some of the Ussing chamber experiments;
Kerry O'Brien, Melissa Levak, Lea Klausli, and Jerod Denton for
valuable technical assistance; and Dr. Alice Givan and Ken Orndorff for
assistance with confocal microscopy. We also thank the anonymous
reviewers for helpful comments on the manuscript and for the suggestion
to conduct dose-response studies with sodium 4-phenylbutyrate.
 |
FOOTNOTES |
These studies were supported by grants from the National Institutes of
Health (DK/HL-45881) and the Cystic Fibrosis Foundation (to B. A. Stanton). J. Loffing was supported by a fellowship from the Swiss
National Science Foundation. Confocal microscopy was performed in the
Herbert C. Englert Cell Analysis Laboratory, which was established by a
grant from the Fannie E. Ripple Foundation and is supported in part by
a Core Grant of the Norris Cotton Cancer Center (CA-23108). B. D. Moyer
was supported by a predoctoral fellowship from the Dolores Zohrab
Liebmann Foundation.
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.
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).
Received 21 October 1998; accepted in final form 17 May 1999.
 |
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