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INTRODUCTION |
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 protein is mutated
and defective in cystic fibrosis (CF), a common lethal genetic disease.
CFTR has been demonstrated to function in two ways: (i) as a protein
kinase A-regulated Cl
channel and (ii) as a regulator of
other membrane conductances (1-3). Thus, CF may not only affect
cAMP-dependent Cl
conductance, but may also
affect other membrane conductances normally regulated by wild-type
CFTR. Such a defect in CFTR-dependent regulation was found
for the epithelial Na+ conductance (1, 4). Thus, enhanced
Na+ conductance was detected in the airways and intestinal
epithelium of CF patients, which is very likely to contribute to
enhanced absorption of electrolytes and water and to the altered
mucociliary clearance as well as intestinal obstructions, respectively
(5-7).
Apart from epithelial Na+ conductance, osmotic water
permeability has been reported to be influenced by CFTR (8, 9). These
studies were performed in Xenopus oocytes and indicated enhanced osmotic cell swelling after expression and
cAMP-dependent activation of CFTR. Since water-injected
control oocytes did not demonstrate such a cAMP-activated water
permeability, the most likely explanation was that cAMP acted through
activation of CFTR. In fact, it was suggested initially that water uses
the same conductive pathway and moves together with Cl
through the CFTR Cl
channel. This pathway should
therefore be formed by transmembrane domains (8). However, it was shown
in a subsequent study (a) that both water and Cl
ions
moved independently when CFTR was activated by an increase in
intracellular cAMP. Independence was demonstrated by showing selective
inhibition of CFTR Cl
conductance by glibenclamide and
selective blockage of water permeability by mercurial compounds and
phloretin (9). In addition, this water pathway activated by CFTR was
permeable for glycerol, too. These results support the assumption that
some sort of endogenous water and glycerol permeability must be present
in Xenopus oocytes and that this is activated by CFTR.
So far, it was unclear whether CFTR-activated water permeability is
unique to Xenopus oocytes or whether a similar interaction can also be observed in mammalian cells. This question was addressed in
this study by measuring cell volume changes and radioactive glycerol
uptake in human airway epithelial and Chinese hamster ovary (CHO)
cells. We found CFTR-dependent activation of osmotic water
permeability in normal respiratory cells and identified one member of
the aquaporin family (AQP3) as the interacting partner. Since
CFTR-dependent stimulation of osmotic water permeability is
absent in airway cells derived from CF patients, we speculate that this has a pathophysiological impact on the lung disease in CF.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human non-CF, CF, and CF airway epithelial
cells transfected with 6REP-wtCFTR have been described in previous
studies (10, 11) and were kindly provided by Dr. D. C. Gruenert
(University of California, San Francisco). CHO-K1 cells and CHO cells
stably expressing wtCFTR (CHO-wtCFTR) or
F508-CFTR (CHO-
F508)
were kindly provided by Dr. X.-B. Chang (Mayo Clinic, Scottsdale, AZ). Cells were cultured on tissue culture plastic dishes or glass coverslips and kept in an atmosphere of 5% CO2 under
conditions described elsewhere (10, 12). CHO cells were transfected
with rat AQP3 cDNA (kindly provided by M. Echevarría,
University of Sevilla, Sevilla, Spain) inserted into expression vector
pZeoSV (Invitrogen) using standard techniques. Isolated colonies were expanded and assayed for AQP3 expression by measurement of glycerol uptake and detection of AQP3 mRNA (RT-PCR).
Cell Volume Measurements by Confocal Microscopy--
Cells grown
on coverslips were loaded with the fluorescent dye calcein/AM (10 µmol/liter for 30 min at room temperature), and repetitive x-z line
scans at 488 nm excitation of one scanning line through three to six
cells were performed using an LSM 410 apparatus (Zeiss, Germany) (13).
A water immersion lens (Zeiss C-APO 63/1.2w) was used to avoid optical
distortions due to refractive index mismatches. The z-line scan
distance was set to result in square voxel; usually these were in
z-steps of 0.33 µm. The size of the confocal pinhole was set to give
a full-width half-maximum in the z-direction of 0.4 µm. The z-line
scans were stacked in a two-dimensional image giving a x-z slide
through the cells. A customized macro allowed the recording and storage
of time series of x-z slides (usually 200-300 images). Cell area and
the mean intensity of the cell area at the cutting z-line were analyzed with Metafluor software (Universal Imaging Corp., West Chester, PA).
Initial area increase and initial intensity decrease induced by
hypotonic bath solution due to omission of mannitol (72.5 mmol/liter NaCl, 0.4 mmol/liter KH2PO4, 1.6 mmol/liter
K2HPO4, 1 mmol/liter MgCl2, 1.3 mmol/liter calcium gluconate, and 5 mmol/liter D-glucose, pH 7.4) were determined in the same cells before and after stimulation and in the presence of IBMX (0.5 mmol/liter) and forskolin (10 µmol/liter).
Radioactive Glycerol Uptake--
The number of cells was
assessed, and cells were incubated in a
[14C]glycerol-containing solution consisting of 160 mmol/liter [14C]glycerol (final activity = 1 mCi/liter or 37 MBq/liter), 65 mmol/liter NaCl, 1.6 mmol/liter
K2HPO4, 0.4 mmol/liter
KH2PO4, 1.3 mmol/liter CaCl2, 1 mmol/liter MgCl2, and 5 mmol/liter D-glucose, pH 7.4, at 37 °C for 1, 2, and 3 min. Cells were rinsed three times
with ice-cold unlabeled glycerol solution and lysed in 100 g/liter SDS
at room temperature. Radioactivity was measured by liquid scintillation
counting. Glycerol uptake was measured in the presence of IBMX (0.5 mmol/liter) and forskolin (10 µmol/liter) and in cells preincubated
with 5 µmol/liter HgCl2 for 15 min.
Expression of AQPs in Airway Epithelial Cells--
Total RNA of
non-CF and CF cells was used for RT-PCR analysis of AQP1, AQP3, AQP4,
and AQP5 expression. The following primers were used: AQP1,
5'-ATGGCCAGCGAGTTCAAGA-3' (sense) and 5'-TAGTAGCCAGCACGCATAG-3' (antisense); AQP3, 5'-ATGGGTCGACAGAAGGAG-3' (sense) and
5'-CTCAGATCTGCTCCTTGT-3' (antisense); AQP4, 5'-ATGGATGCTGAGGTGCCA-3'
(sense) and 5'-CACACACTCTCCATCTCC-3' (antisense); and AQP5,
5'-ATGAAGAAGGAGGTGTGC-3' (sense) and 5'-TCATCTCCAGGGAGCCAG-3' (antisense). The 879-base pair coding sequence of human AQP3 was RT-PCR-amplified from non-CF cells using 5'-CCGCCATGGGTCGACA-3' (sense)
and 5'-CTCAGATCTGCTCCTTGT-3' (antisense). Resulting PCR products were
sequenced (Applied Biosystems Model 373A). For expression in
Xenopus oocytes, cDNA encoding human AQP3 was subcloned
into oocyte expression vector pTLN, which uses the Xenopus
-globin untranslated regions to boost expression in oocytes (14).
Airway cells and CHO cells grown on glass coverslips were fixed with methanol/acetone/Formalin solution (45:45:5) for 90 s. After
washing with TBS-P (1 liter of 0.9 g of Tris base, 6.85 g of
Tris-HCl, 8.78 g of NaCl, and 0.1% Tween 20, pH 7.5), cells were
incubated with antibodies against AQP3 (1 µg/ml; kindly provided by
M. A. Knepper, National Institutes of Health, Bethesda, MD) for 30 min at room temperature. All antibodies were diluted in RPMI 1640 medium (Sigma R 1145) with 10% heat-inactivated bovine serum and 0.1%
sodium acid. After washing with TBS-P, cells were incubated with mouse
anti-rabbit IgG (1:50; Dako M0737) for 30 min, washed with TBS-P,
incubated with rabbit anti-mouse IgG (1:25; Dako Z0259) for 30 min,
washed again, and incubated with APAAP complex (1:50; Dako D0651) for
30 min. Peroxidase reaction was visualized by naphthol AS-BI phosphate
(Sigma N 2250) and new fuchsin (Merck). The reaction was stopped
with double-distilled H2O, and cells were
counterstained with Mayer's hematoxylin solution and embedded in
Kaiser's glycerol gelatin.
Antisense Oligonucleotides--
Subconfluent (70%) non-CF and
CF-wtCFTR cells were incubated with 20 µmol/liter phosphorothioated
sense and antisense oligonucleotides (AQP3, 5'-CAGCTCCTTCTGTCGACCCAT-3'
(antisense) and 5'-ATGGGTCGACAGAAGGAGCTG-3' (sense); AQP1,
5'-CTTCTTGAACTCGCTGGCCAT-3' (antisense); and AQP5, 5'-GGAGCACACCTCCTTCTT-CAT-3' (antisense)) in serum-free culture medium
supplemented with 2% Ultroser G (Life Technologies, Inc.) for 48 h, and subsequently, glycerol uptake was measured.
Osmotic Water and Glycerol Permeability in Xenopus
Oocytes--
Isolation and microinjection of oocytes have been
described in a previous report (15). Oocytes of identical batches were injected with 10-50 ng of cRNA (wild-type CFTR, G551D-CFTR, and hAQP3
cloned from human airway cells). Water-injected oocytes served as
controls. The osmotic water permeability coefficient (Pf) was calculated from the rate of hypotonic
volume increase, measured by gravimetric techniques at 22 °C (9). Hypotonicity was induced by omission of 120 mmol/liter mannitol from
ND96 normotonic medium. Pf was calculated according to Ref. 16: Pf = dV/dt·[1/S·
], with
S being the oocyte surface area, V being the
volume, and 
being the osmolality gradient at zero time. The
ratio dV/dt was calculated from the weight change
1 min after exposure to hypotonic buffer. Glycerol permeability
(Pgly) was calculated from the initial rate of
[14C]glycerol uptake per oocyte during incubation with
[14C]glycerol (2 µCi/ml) and 1 mmol/liter glycerol.
Oocytes were lysed overnight in 100 g/liter SDS at room temperature,
and radioactivity was counted.
Compounds and Statistics--
All chemicals used were the
highest grade of purity available. Data are given as means ± S.E.
(n), where n refers to the number of experiments.
Paired and unpaired t tests were used for statistics with a
p value of <0.05, indicating statistical significance.
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RESULTS |
Water and Glycerol Permeability Is Increased by cAMP in Normal
Airway Cells, but Not in Cells Derived from CF Patients--
Water
permeability in airway epithelial cells was assessed by measuring
osmotically induced cell swelling using the novel microscopic
techniques described above (13). Rapid change to hypotonic bath
solution resulted in an increase in cell area and a decrease in
fluorescence intensity, as shown for a non-CF cell in Fig.
1. Linear regression was fitted to the
data points obtained during the first 30 s after exposure to the
hypotonic bath solution (Fig. 1B). Stimulation with IBMX and
forskolin augmented the area increase and the fluorescence pixel
(calcein concentration) decrease elicited by hypotonic bath solution.
These measured volume changes necessarily underestimate the real rate
of hypotonic cell swelling and the effects of CFTR on osmotic water
permeability because the cells activate their volume regulatory
decrease mechanisms, which will be even augmented when CFTR
Cl
conductance is activated (9). The summary shown in
Fig. 2A indicates that cell
swelling was significantly enhanced after activation of CFTR
(solid bars). Such a cAMP-dependent increase in
hypotonic cell swelling was not observed in airway epithelial cells
derived from CF patients (Fig. 2B).

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Fig. 1.
Cell volume measurements by confocal
microscopy. A, non-CF airway epithelial cells were
loaded with calcein/AM, and images from a confocal x-z line scan were
recorded from two cells. Cell area and fluorescence intensity are taken
as independent quantitative measures of the cell volume. Cell swelling
in response to a rapid change to hypotonic bath solution
(Hypo) resulted in an increased cell area and a decrease in
fluorescence intensity, coded in false colors. B, initial
and absolute area increase and intensity decrease induced by hypotonic
bath solution were determined in the same cell under control conditions
and after stimulation with IBMX (0.5 mmol/liter) and forskolin (10 µmol/liter). Initial change of area and intensity was calculated by
linear regression of the first 30 s of recording in hypotonic
solution.
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Fig. 2.
Activation of CFTR enhances water
permeability in non-CF but not CF airway epithelial cells.
A, shown is a summary of cell volume measurements in non-CF
cells. Stimulation by IBMX and forskolin (black bars)
significantly enhanced initial area increase and initial fluorescence
decrease in cells compared with control conditions before and after
stimulation (n = 18). CFTR-activated water permeability
led to an increase in the absolute area, whereas fluorescence intensity
was decreased (n = 18). B, in CF epithelial
cells, stimulation by IBMX and forskolin had no effect on cell volume
change (n = 12). Data are means ± S.E.
Asterisks indicate significant difference from control
(p < 0.05).
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Previous studies have demonstrated that osmotic water permeability
activated by CFTR in Xenopus oocytes is also permeable for
glycerol. Therefore, we measured [14C]glycerol uptake in
airway epithelial cells that expressed endogenous wild-type CFTR
(non-CF), mutant CFTR (CF), or exogenous wild-type CFTR (CF-wtCFTR)
(Fig. 3). After stimulation of non-CF
cells with IBMX and forskolin, [14C]glycerol uptake
(closed circles) was enhanced when compared with control
uptake (open circles). Such an activation of
[14C]glycerol uptake by cAMP was not observed in CF
cells. Here, [14C]glycerol uptake was even attenuated
when the cAMP-dependent pathway was activated. However,
expression of exogenous wild-type CFTR in CF cells (CF-wtCFTR) restored
the effects of IBMX and forskolin. Pretreatment of the cells with
HgCl2 (5 µmol/liter, 15 min) completely inhibited the
effect of cAMP on [14C]glycerol uptake in non-CF cells as
well as in CF cells expressing exogenous CFTR (closed
squares). At this concentration, CFTR Cl
currents
were not affected (data not shown). These results suggest, in agreement
with what has been observed in Xenopus oocytes (9), that,
also in human airway epithelial cells, a glycerol-permeable water
channel is activated by CFTR.

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Fig. 3.
Activation of CFTR increases glycerol
permeability. [14C]Glycerol uptake was measured in
the initial 3 min after exposure (open circles) in non-CF
and CF airway epithelial cells and in transfected CF cells (CF-wtCFTR).
Stimulation of non-CF cells with IBMX and forskolin increased
[14C]glycerol uptake, whereas a decrease in
[14C]glycerol uptake was observed for CF cells
(closed circles) (n = 3 each).
cAMP-dependent inhibition of glycerol uptake in CF cells is
currently poorly understood and is due to another CFTR-independent
mechanism. Transfection with wtCFTR (CF-wtCFTR) restored the effects of
IBMX and forskolin on [14C]glycerol uptake in CF cells
(n = 6). Pretreatment of non-CF and CF-wtCFTR cells
with HgCl2 (5 µmol/liter, 15 min) abolished
forskolin/IBMX-induced [14C]glycerol uptake (closed
squares). Data are means ± S.E. Asterisks
indicate significant difference from control (p < 0.05).
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AQP3 Is Responsible for CFTR-dependent Glycerol
Uptake--
Water permeability in mammals is facilitated in many cells
by specific water channels, so-called aquaporins (17). Expression of
various types (AQP1, AQP3, AQP4, and AQP5) in non-CF and CF airway
epithelial cells was performed by RT-PCR. No expression of AQP4 could
be detected in airway epithelial cells. However, expression of AQP1,
AQP3, and AQP5 was found (data not shown). Message for AQP3 was
detected in freshly isolated airway epithelial cells from nasal polyps
as well as in various cultured CF and non-CF airway epithelial cells
(Fig. 4B). Since AQP3, in
contrast to AQP1 or AQP5, has been described to transport glycerol (18, 19), these results suggest that AQP3 is the water pathway responsible for CFTR-dependent water and glycerol uptake. This was
further supported by staining of AQP3 in these cells.
Immunocytochemical localization of AQP3 demonstrated abundant
expression of this protein throughout the plasma membrane (Fig.
4A). AQP3 was also immunolocalized in CHO cells expressing
AQP3 and in CF airway cells, but not in control CHO cells or in the
absence of the primary antibody (Fig. 4A).

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Fig. 4.
Expression of AQP3 and AQP3 mRNAs in
human airway epithelial cells. A, immunocytochemical
localization of AQP3 in non-CF airway epithelial cells. Cells were
labeled with antibodies against AQP3 and visualized using APAAP complex
(upper panel). Control staining was carried out without
primary antibody (lower panel). Bars = 30 µm. B, total RNA of airway cells derived from human nasal
polyps and cultured non-CF and CF airway epithelial cells as well as
cultured CF airway cells transfected with wtCFTR was
reverse-transcribed, and expression of AQP3 was detected by PCR
amplification. RT± indicates PCR of RNA samples that were
reverse-transcribed (+) or that were used without previous reverse
transcription (control) ( ). Experiments were performed in
triplicates. bp, base pairs.
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Furthermore, normal respiratory epithelial cells expressing endogenous
CFTR (non-CF) and CF epithelial cells expressing exogenous wild-type
CFTR (CF-wtCFTR) were incubated with AQP3 sense and antisense
oligonucleotides, respectively. After 48 h of incubation, CFTR-dependent, i.e. forskolin/IBMX-induced,
glycerol uptake was abolished in non-CF and CF-wtCFTR cells. No such
effects on forskolin/IBMX-activated glycerol uptake were observed in
cells incubated with sense oligonucleotides for AQP3 or antisense
oligonucleotides for AQP1 and AQP5 (Fig. 5), suggesting that AQP3 is responsible
for CFTR-dependent activation of osmotic water permeability
in airway epithelial cells.

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Fig. 5.
cAMP-dependent activation of
glycerol uptake is abolished in AQP3 antisense oligonucleotide-treated
non-CF cells and in CF cells expressing wtCFTR (CF-wtCFTR). After
48 h of treatment with antisense oligonucleotides for AQP3,
CFTR-activated glycerol uptake was abolished. Incubation with AQP1 or
AQP5 antisense oligonucleotides or AQP3 sense oligonucleotides was
without any significant effects. Data are expressed as means ± S.E. (n = number of measurements). Asterisks
indicate significant difference from control (p < 0.05).
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CFTR Is Required for Activation of AQP3 by cAMP in Xenopus
Oocytes--
Human AQP3 was cloned from human airway epithelial cells
used here by RT-PCR. The obtained nucleotide coding sequence was identical to the sequence published in a previous report (20). Expression of the cloned hAQP3 cDNA in Xenopus oocytes
induced an enhanced osmotic glycerol permeability coefficient
(Pgly) (9) (11.0 ± 1.2 × 10
6 cm/s; n = 13) compared with control
oocytes (1.7 ± 1.7 × 10
6 cm/s;
n = 11). When CFTR and hAQP3 were coinjected into
Xenopus oocytes, enhanced basal Pgly
was further augmented by stimulation of the oocytes with IBMX (Fig.
6C). No increase was observed
when hAQP3 was expressed alone (data not shown). Moreover, in
hAQP3/CFTR-coexpressing oocytes, activation of CFTR by IBMX in the
presence of an isotonic bath solution induced initial cell shrinkage
due to Cl
exit from the oocytes (Fig. 6A).
Subsequent exposure to an extracellular hypotonic solution in the
continued presence of IBMX augmented cell swelling and increased the
osmotic water permeability coefficient (Pf)
significantly, indicating activation of hAQP3 water channels by CFTR
(Fig. 6, A and C). As described for the
experiments performed in airway cells, the detected volume changes will
even underestimate the impact of CFTR on osmotic water permeability because of the volume regulatory decrease that is augmented by activation of CFTR Cl
conductance (9). A mutation in the
first nucleotide-binding fold of CFTR (G551D-CFTR) abolished the
effects of IBMX on Pf (Fig. 6B),
confirming impaired regulation of hAQP3 by mutant forms of CFTR.

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Fig. 6.
wtCFTR activates human AQP3 in
Xenopus oocytes. A, shown is the
volume increase in oocytes under hypotonic conditions as measured by
gravimetric techniques (9). Hypotonic (Hypo) volume changes
in water-injected oocytes were independent of the presence of IBMX
(open circles; n = 9). Hypotonic cell
swelling was augmented by IBMX in AQP3/CFTR-coexpressing oocytes
(closed circles; n = 8). After initial
osmotic swelling, cells were allowed to recover in isotonic bath and
were then exposed to forskolin/IBMX, which led to cell shrinkage due to
activation of CFTR Cl conductance and Cl
exit. B, the calculated osmotic water permeability
coefficient (Pf) in Xenopus oocytes was
larger after stimulation with IBMX due to CFTR-dependent
activation of the endogenous water channel in Xenopus
oocytes (9). Pf was increased in
AQP3/CFTR-coexpressing oocytes and was further enhanced by IBMX,
whereas oocytes coexpressing AQP3 and G551D-CFTR did not show an
increase in Pf upon stimulation by IBMX.
C, the osmotic glycerol permeability coefficient
(Pgly) (9) was enhanced by IBMX in
CFTR-expressing oocytes. In hAQP3/wtCFTR-coexpressing oocytes,
base-line Pgly was enhanced and was further
increased by IBMX. Data are means ± S.E. (n).
Asterisks indicate significant difference from control
(p < 0.05).
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Only CHO Cells Coexpressing Both CFTR and Rat AQP3 Demonstrate
cAMP-activated [14C]Glycerol Uptake--
The above
results obtained in Xenopus oocytes have been verified in a
mammalian expression system by coexpressing rAQP3 and wtCFTR or
F508-CFTR, respectively, in CHO cells.
[14C] Glycerol uptake was measured before and after
stimulation with forskolin and IBMX. As shown in Fig.
7, control cells or cells expressing only
wtCFTR or
F508-CFTR demonstrated base-line
[14C]glycerol uptake, which was not influenced by
stimulation with forskolin and IBMX. Control,
F508-CFTR-, and
wtCFTR-expressing CHO cells were transfected with rAQP3, and expression
was detected by RT-PCR analysis (data not shown). Additional expression
of rAQP3 enhanced base-line glycerol uptake in all three cells lines to
different degrees. Control and
F508-CFTR-expressing cells did not
further enhance glycerol uptake upon exposure to IBMX and forskolin,
whereas it was significantly increased in wtCFTR-expressing cells (Fig.
7). Both the basal [14C]glycerol uptake caused by
expression of rAQP3 and the uptake caused by activation of wtCFTR were
inhibited by HgCl2 (5 µmol/liter). These experiments
clearly indicate CFTR-dependent activation of hAQP3 when
both proteins are expressed heterologously in CHO cells.

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Fig. 7.
wtCFTR activates rAQP3 in CHO cells. The
variable endogenous [14C]glycerol uptake in CHO cells
(control) and in CHO cells expressing either wtCFTR or F508-CFTR
remained unchanged upon stimulation by IBMX and forskolin. Additional
expression of rAQP3 resulted in a significant increase in
[14C]glycerol uptake in all three cell lines. When
stimulated by IBMX and forskolin, only cells coexpressing wtCFTR and
rAQP3 demonstrated a further increase in glycerol uptake, indicating
the requirement of both wtCFTR and rAQP3. The
CFTR-dependent increase in [14C]glycerol
uptake was inhibited by HgCl2 (5 µmol/liter). Data are
means ± S.E. (n). Asterisks indicate
significant difference from control (p < 0.05).
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DISCUSSION |
The present data show compelling evidence for a novel type of
CFTR-dependent regulation of yet another epithelial
membrane permeability. They demonstrate that CFTR, when stimulated by
protein kinase A, activates a water permeability in respiratory
epithelial cells. These results suggest coupling between
Cl
transport as performed by the CFTR Cl
channel and water transport, thereby adjusting water permeability to
electrolyte transport in the airway epithelium. In that sense, this
mechanism would help to secure proper hydration of the surface fluid as
well as mucociliary clearance (21, 22).
Several different types of aquaporins are expressed in the airways,
including AQP1, AQP3, AQP4, and AQP5 (23). AQP expression in the lung
shows a complex pattern suggesting functional specialization of the
different AQPs, which seems to be required in the postnatal period as
well as in the adult lung (23). AQP3 is expressed primarily in
basolateral membranes of acinar cells forming the submucosal glands
(24). These glands are regarded as the predominant site for electrolyte
secretion (24-26). On the other hand, AQP3 is expressed abundantly in
nonpolarized basal cells of the trachea and in basolateral membranes of
nasal surface epithelial cells (23, 24) involved in reabsorption of
electrolytes (6, 21). In this study, expression of AQP3 and its
CFTR-dependent regulation were detected in cultured airway
epithelial cells, which demonstrate features of cell differentiation
such as expression of amiloride-sensitive Na+ currents (10,
27). However, expression of AQP3 does not seem to be limited to the
basolateral membranes of these cells, thereby reflecting the properties
of basal cells rather those that of surface cells. Within the
respiratory tract, CFTR is expressed predominantly in apical membranes
of submucosal gland cells and, to a lesser degree, also in those of
surface epithelial cells. CFTR Cl
currents can also be
detected in basal cells (28), but there is no evidence for CFTR
expression in basolateral membranes of either cell type (29). This
suggests that, except for the basal cells, CFTR and AQP3 are expressed
on the different poles of airway cells. We may therefore currently only
speculate in what cell type of the highly differentiated respiratory
tract the CFTR-AQP3 interaction actually takes place. Further studies
have to clarify whether AQP3 contributes to both airway absorption and
secretion and how it participates in formation of the airway surface
liquid, which is essential for proper mucociliary clearance.
CFTR-dependent activation of AQP3 does not take place in
airway cells carrying the CF defect or in cells overexpressing both
AQP3 and mutant forms of CFTR. We may therefore speculate that
regulation of AQP3 by CFTR is impaired in the respiratory tract of CF
patients, which may contribute to the imbalance between secretion and
absorption detected in CF airways (6, 26, 30).
Activation of AQP3 by CFTR is yet another example for
CFTR-dependent regulation of other epithelial membrane
conductances. Several other ion conductances have been reported to be
regulated by CFTR, like the epithelial Na+ conductance and
intermediate conductance Cl
channel (outwardly rectified
Cl
channel and intermediate conductance outwardly rectifying
chloride channel) (1, 4, 31). Furthermore, the affinity of
K+ channels like Kir6.1 and ROMK2 for the inhibitory
compound glibenclamide is enhanced by CFTR (32). The mechanisms of this
regulation are currently under examination (3). They are probably
examined in most detail for CFTR-dependent inhibition of
the epithelial Na+ conductance, which was shown only
recently to take place in the native respiratory tissue (7).
CFTR-dependent regulation of epithelial Na+
conductance depends on the presence of Cl
ions and the
existence of an intact first nucleotide-binding domain of CFTR. It
takes place even in isolated membrane patches, suggesting a rather
direct interaction of both proteins (33-36). Although the actual
mechanism of this interaction still remains obscure, results from these
studies may also guide further experiments to unmask
CFTR-dependent regulation of AQP3. The results shown here
add a new example to the growing list of CFTR-dependent
interactions with other membrane proteins, thereby emphasizing the
function of CFTR as a conductance regulator. The results may also help to understand the complex pattern of the common disease cystic fibrosis.