Institut National de la Santé et de la Recherche Médicale, 1 Unité 478 et 2 Unité 426, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, 75870 Paris, France
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ABSTRACT |
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The role of
the cystic fibrosis transmembrane conductance regulator (CFTR) in the
renal cortical collecting duct (CCD) has not yet been fully elucidated.
Here, we investigated the effects of deamino-8-D-arginine
vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in
primary cultured CCDs microdissected from normal [CFTR(+/+)] and
CFTR-knockout [CFTR(/
)] mice. dDAVP stimulated the benzamyl
amiloride (BAm)-sensitive transport of Na+ assessed by the
short-circuit current (Isc) method in both
CFTR(+/+) and CFTR(
/
) CCDs to a very similar degree. Apical
addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or
glibenclamide partially inhibited the rise in
Isc induced by dDAVP and ISO in BAm-treated
CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter
Isc in BAm-treated CFTR(
/
) CCDs. dDAVP
stimulated the apical-to-basal flux and, to a lesser extent, the
basal-to-apical flux of 36Cl
in CFTR(+/+)
CCDs. dDAVP also increased the apical-to-basal
36Cl
flux in CFTR(
/
) CCDs but not the
basal-to-apical 36Cl
flux. These results
demonstrate that CFTR mediates the cAMP-stimulated component of
secreted Cl
in mouse CCD.
cyclic adenosine monophosphate; kidney; epithelial sodium channel; cystic fibrosis transmembrane conductance regulator; cystic fibrosis
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INTRODUCTION |
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THE ROLE OF THE CYSTIC
FIBROSIS transmembrane conductance regulator (CFTR) in the kidney
remains uncertain, because there is no major disruption of renal
function in cystic fibrosis (CF) patients (29). The CFTR
protein has been detected in most segments of the renal tubule,
although its exact cellular and/or membranous localization remains to
be identified. CFTR-like Cl currents and/or
CFTR mRNA have been reported in the amphibian A6 cells
(19, 34) and in a variety of cultured cells from the
distal convoluted tubule (22), cortical collecting duct (CCD) (8, 20), and inner medullary collecting duct (IMCD) (7, 14, 33). It is now generally agreed that CFTR
is expressed mainly at the apical surface of distal and collecting duct
cells, and there is increasing indirect evidence that renal
Cl
secretion, a minor process under most physiological
circumstances, is one function of CFTR in the terminal parts of the
renal tubule, in particular in the IMCD (14, 33).
Because CFTR can act as a downregulator of the amiloride-sensitive
epithelial sodium channel (ENaC) (3, 30, 31), which is
normally expressed in principal collecting duct cells (6), it has been suggested that cAMP may stimulate CFTR-like
Cl channels and inhibit ENaC in cultured mouse M-1 CCD
cells (17). More recently, Kibble et al. (15)
have shown that
508-CFTR transgenic mice exhibited normal urinary
Na+ excretion under salt repletion and chronic salt
depletion but that the fractional Na+ excretion induced by
amiloride in mice subjected to salt restriction is greater in CF than
in control animals. Na+ absorption mediated by ENaC may
therefore be increased in CF CCDs, at least under particular
physiological circumstances.
Nagy et al. (20) have previously shown that vasopressin
(AVP), 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP), and
isoproterenol (ISO) all stimulate a
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive
Cl conductance in primary cultures of immunoselected
rabbit CCD cells. These authors suggested that, in intercalated cells,
CFTR may underlie at least part of this Cl
conductance.
CFTR mRNA has been shown to be expressed mainly in
intercalated cells and to a lesser extent in principal cells (32). CFTR and ENaC have also been
shown to be expressed in the immortalized mouse M-1 CCD cells
(17) and mouse principal mpkCCDcl4 cells
(unpublished results). The questions therefore arise as to
whether the stimulation of Na+ transport mediated by ENaC
is accompanied by the secretion of Cl
into the CCD and to
what extent CFTR may regulate ENaC activity in the
CCD. To address these questions, we used
cftrm1unc mice (28), which lack
CFTR-mediated, cAMP-dependent Cl
secretion in the colon,
airways, and exocrine pancreas cells (5). We used the
short-circuit current (Isc) method and
36Cl
flux studies to carry out ion transport
studies with the use of primary cultures of CCD cells microdissected
from the kidneys of wild-type [CFTR(+/+)] and CFTR-knockout mice
[CFTR(
/
)]. We investigated the effects of
deamino-8-D-arginine vasopressin (dDAVP), an analog that
binds to the V2 receptors and mimics the effects of
vasopressin on Na+ absorption in principal cells
(23), and of ISO, which is known to stimulate cAMP only in
intercalated cells (9).
The data from this study show that the component of secreted
Cl stimulated by dDAVP or ISO detected in wild-type
CFTR(+/+) CCD cells was absent in CFTR(
/
) CCD cells. They also show
that CFTR had almost no inhibitory action on dDAVP-stimulated
Na+ absorption mediated by ENaC in this model of mouse CCD
cells in primary culture.
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MATERIALS AND METHODS |
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Cell culture.
Experiments were conducted on male cftrm1unc
mice (28) bred at the Centre de Développement des
Techniques Avancées pour l'Expérimentation Animale
(Orleans, France). This strain of mice was originally derived from
ES129/Sv cells injected into C57BL/6 embryos. Mice were back-crossed
with C57BL/6 mice for three generations and then intercrossed. The 5- to 8-wk-old wild-type CFTR(+/+) mice (n = 21) and
CFTR(/
) mice homozygous for the disrupted CFTR gene
(n = 16) were killed by cervical dislocation, and their
kidneys were removed. CCDs were microdissected out under sterile
conditons. Isolated CCDs (8-10 fragments, 0.2-0.5 mm long)
were seeded on Transwell permeable filters (0.4-µm pore size, 0.33 cm2 insert growth area; Corning Costar, Cambridge, MA) and
grown in a defined medium [DM (DMEM): Ham's F-12 (1:1 vol/vol), 60 nM sodium selenate, 5 µg/ml transferrin, 2 mM glutamine, 50 nM
dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor
(EGF), 5 µg/ml insulin, 2% fetal calf serum (FCS), 20 mM HEPES, pH
7.4] at 37°C in a 5% C02-95% air atmosphere. After the
first 5 days, the medium was changed every 2 days. Experiments were
carried out 2 wk after seeding, by use of confluent cells that had
developed high transepithelial electrical resistance (>700
· cm2). All experiments were performed in
accordance with the guidelines of the French Agricultural Office and in
compliance with the legislation governing animal studies.
Electronmicroscopy and immunohistochemical studies. Confluent cells grown on permeable filters were fixed for 2 h with 0.2% glutaraldehyde in 0.1 M cacodylate buffer, embedded in epon, and processed for electronmicroscopy. Cells were also fixed in ice-cold methanol and processed for immunofluorescence using biotinylated Dolichos biflorus agglutinin (DBA; Vector, Burlingame, CA), as previously described (1). Specimens were examined under a Zeiss microscope and photographed.
RNA extraction and RT-PCR.
Total RNA was extracted from microdissected CFTR(+/+) and CFTR(/
)
CCDs by use of the RNA-PLUS extraction kit (Bioprobe Systems, Montreuil-sous-Bois, France) and reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Eragny,
France) at 42°C for 45 min. cDNA and non-reverse-transcribed RNA were
amplified for 28-38 cycles in a total volume of 100 µl of PCR
buffer (50 mM KCl, 20 mM Tris · HCl, pH 8.4) containing 40 µM
dNTP, 1.5 mM MgCl2, 1 µCi [
-32P]dCTP
(NEN, Le Blanc Mesnil, France), 1 U Taq polymerase, 29 pmol
of
-ENaC primers (13), and 1.5 pmol of
-actin primers (8). The CFTR mRNA
was also analyzed by RT-PCR with the use of 42 pmol of CFTR
primers from the mouse CFTR exon 9 and exon 13, as described
by Marvão et al. (18), and with 0.5 pmol of
-actin primers. The thermal cycling program was as
follows: 94°C for 30 s, 54°C (for
-ENaC) or
60°C (for CFTR) for 30 s, and 72°C for 60 s.
Amplification products were run on 4% polyacrylamide gels and
autoradiographed. The sets of CFTR and ENaC
primers used yielded amplified products of the expected size, and
sequence analysis of PCR products confirmed that they matched the
expected cDNA analyses (data not shown).
Electrophysiological studies.
Confluent CFTR(+/+) and (/
) CCD cells were grown on
0.33-cm2 Transwell filters (Corning Costar) in DM medium
until confluent (day 14), and then in DM containing no EGF,
hormones, FCS, or HEPES [Ham's F-12 medium (HFM) containing 29 mM
NaHCO3] for the final 2 h. The filters were
mounted in a modified Ussing-type chamber (Diffusion Chamber System,
Costar) connected to a voltage clamp apparatus via glass barrel
Micro-Reference Ag/AgCl electrodes. Cell layers were bathed on both
sides (0.2 ml for the apical side and 0.6 ml for the basal side) with
HFM warmed to 37°C and continuously gassed with 95%
02-5% C02 to keep the pH at 7.4. Isc (µA/cm2) was measured by
clamping the open-circuit transepithelial voltage (VT) to 0 mV for 1 s. By convention, a
positive Isc value corresponded to a flow of
positive charges from the apical to the basal solution.
Apical Na+ substitution
experiments.
Isc was measured on CFTR(+/+) and (/
) CCD
cells in which the apical NaHCO3-HFM medium was replaced by
a Na+-free solution (in mM: 156 N-methyl-D-glucamine, 4 KCl, 0.7 MgCl2, 0.4 MgSO4, 1.05 CaCl2, 20 glucose, 8 HEPES, pH 7.4) (8). The basal side of the
filter was bathed with the HFM warmed to 37°C and continuously gassed
with 95% 02-5% CO2, and
Isc was measured after a 1-h equilibration
period. For all experiments, dDAVP (Ferring Pharmaceutical, Malmo,
Sweden) or ISO (Sigma, St. Louis, MO) was added to the basal side of
the filters. Benzamyl amiloride (BAm; Sigma),
5-nitro-2-(3-phenylpropamino)benzoate (NPPB; Research Biochemicals, Natick, MA), glibenclamide (Glb; Sigma) or DIDS (Sigma) was added to the apical side of the filters. These agents were dissolved at 0.1 M in DMSO, and diluted 1:1,000 (10
4
M final concentration for NPPB, Glb, and DIDS) or 1:100,000
(10
6 M final concentration for BAm) in appropriate medium.
36Cl flux studies.
Transepithelial fluxes of 36Cl
(Amersham, Les
Ulis, France) were measured on confluent cells grown on Transwell
filters (0.33-cm2 insert growth area). The inside of the
filters was filled with 150 µl, and the outside was immersed in 600 µl of NaHCO3-HFM. 36Cl
(400 nCi/ml) was added to the apical or basal medium bathing the cells. Cell
layers were incubated at 37°C, and 50-µl samples of apical or basal
medium were collected from opposite sides of the filters 60 min after
the radioactive tracers were added. The radioactivity was measured in a
liquid scintillation counter (LKB; Pharmacia). In parallel,
VT and transepithelial electrical resistance (RT) were monitored using the Millicell
electrical resistance clamp apparatus (Precision Instrument Design,
Tahoe City, CA) as previously described (1, 8).
Statistical analysis. Results are expressed as means ± SE from (n) experiments. Significant differences between groups were analyzed by unpaired or paired Student's t-test. A P value <0.05 was considered significant.
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RESULTS |
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ENaC and CFTR mRNAs in isolated CCDs and the morphology of primary
cultured CCD cells.
ENaC was detected by RT-PCR using -ENaC
specific primers (13) in both CFTR(+/+) and CFTR(
/
)
microdissected CCDs (Fig. 1). CFTR
transcripts, analyzed by RT-PCR using primers from the mouse
CFTR exon 9 and exon 13 (18), were detected in
microdissected CFTR(+/+) CCDs, but not in CFTR(
/
) CCDs (Fig. 1).
CFTR(+/+) and CFTR(
/
) CCDs grown to confluence on permeable filters
formed monolayers of cuboid cells (Fig.
2A). Both CFTR(+/+) and
CFTR(
/
) CCD cells in primary culture exhibited a similar
heterogeneous pattern of lectin DBA binding (Fig. 2B),
suggesting that, as intact CCDs, they were composed of both
intercalated and principal cells (12). The
electrophysiological parameters also showed that CCDs seeded on
permeable filters had the typical electrophysiological features of
tight epithelia. Confluent CFTR(+/+) and CFTR(
/
) CCD cells seeded
and grown on 0.33-cm2 permeable filters (day 14 after seeding) exhibited almost identical high
RT, negative VT, and
positive Isc under basal conditions (Table
1). Taken together, these results
indicate that cultured CCDs microdissected from wild-type and
CFTR-knockout mice and grown on filters formed confluent layers of
tight epithelial cells suitable for electrophysiological studies.
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Effect of Na+ and Cl
channel inhibitors on dDAVP-stimulated Isc in primary
cultures of CFTR(+/+) and CFTR(
/
) CCDs.
The effect of dDAVP, which acts on ion transport via a
cAMP-dependent pathway, was tested on confluent cultured CFTR(+/+) and CFTR(
/
) CCD cells grown on filters. All subsequent experiments were performed using 10
8 M dDAVP, a concentration shown
to give greatest rise in Isc in cultured mouse
CCD cells (8). As shown in Fig.
3, the rise in Isc
caused by dDAVP after 20 min was slightly, but not significantly, lower
in the CFTR(+/+) CCD cells (50.6 ± 10 µA/cm2,
n = 17) than in the CFTR(
/
) CCD cells (61.8 ± 12.1 µA/cm2, n = 16). In both cases, the
apical addition of BAm (10
6 M), a potent ENaC channel
blocking agent (16), almost completely inhibited the
dDAVP-stimulated Isc after 10 min (Fig. 3). As a result, the BAm-sensitive component of Isc was
slightly, but not significantly, greater in CFTR(
/
) CCD than in
CFTR(+/+) CCD cells (Fig. 3). Thus, under our experimental conditions
and by using symetrical high Na+ solutions (i.e., HFM),
ENaC-mediated Na+ absorption was not statistically
significantly increased in primary cultured mouse CFTR(
/
) CCDs
expressing ENaC but lacking functional CFTR
compared with cultured wild-type CCDs coexpressing CFTR and ENaC.
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ISO-stimulated Isc in primary cultures of
CFTR(+/+) CCDs.
In the absence of reliable anti-CFTR antibodies suitable for
immunofluorescence studies on mouse kidney sections or cultured CCD
cells, the exact intrarenal and cellular distribution of the CFTR
protein in the mouse kidney still remains to be determined. Our results
strongly suggest that the concomitant increase in Na+
absorption mediated by ENaC and the electrogenic secretion of Cl mediated by CFTR induced by dDAVP both occur in
principal cells. However, it has been shown that CFTR mRNA
is also expressed in intercalated cells (32). We therefore
tried to find out whether ISO (10
5 M), a
-adrenergic
agonist known to stimulate cAMP in intercalated cells (9),
would also increase the secretion of Cl
without affecting
Na+ absorption in cultured mouse CCD cells.
Isc recordings were first performed with
symetrical, high Na+ levels (i.e., HFM) to find out whether
ISO affected Isc in the absence of BAm. Basal
addition of ISO increased Isc in CFTR(+/+) CCD
cells (Fig. 6A). Results from
individual monolayers showed that the Isc was
always significantly greater in ISO-treated than in untreated CFTR(+/+)
CCD cells (Fig. 6B). Furthermore, the BAm-sensitive Isc, which reflected ENaC-mediated
Na+ absorption, remained unchanged in untreated and
ISO-treated CFTR(+/+) CCD cells (Fig. 6C).
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dDAVP- and ISO-stimulated Isc in primary cultures of
CFTR(+/+) and CFTR(/
) CCDs
measured in the absence of apical Na+.
Apical Na+ replacement experiments were undertaken to
clarify the contribution of CFTR in the cAMP-dependent secretion of
Cl
in cultured mouse CCD cells incubated either with
dDAVP or ISO. Because the symmetrical (apical plus basal) replacement
of Na+ by
N-methyl-D-glucamine-supplemented solutions did
not permit sufficiently stable and reproducible
Isc recordings, experiments were performed on
primary-cultured CCD cells that were bathed on the apical side with the
Na+-free solution, while the basal side of the filter was
bathed with the HFM, as described in MATERIALS AND METHODS.
Under these experimental conditions, Isc
significantly fell by 74% in the untreated cultured CFTR(+/+) CCDs and
by 63% in the CFTR(
/
) CCD cells. The possibility could not still
be excluded that some HFM containing Na+ remained in the
apical compartment after washing and replacement of the apical (200 µl) HFM medium by the Na+-free solution. Therefore, BAm
(10
6 M) was once again added to the apical
Na+-free solution. BAm further reduced the
Isc (by ~4-5 µA/cm2) in
both CFTR(+/+)and CFTR(
/
) CCD cells. Under these experimental conditions, the subsequent addition of 10
8 M dDAVP still
induced a rise in Isc in CFTR(+/+) CCDs that was inhibited by 41% after the apical addition of NPPB (Fig.
7, top). This confirmed that
dDAVP does stimulate the secretion of Cl
in wild-type
cultured mouse CCDs. In contrast, dDAVP did not induce any significant
change in the Isc recorded under apical Na+-free condition in cultured CFTR(
/
) CCDs (Fig.
7, top). Taken together, these results provide further
evidence that CFTR mediates the dDAVP-stimulated secretion of
Cl
in this model of cultured mouse CCDs.
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Effect of dDAVP on Cl flux in primary cultures of
CFTR(+/+) and CFTR(
/
) CCDs.
Unidirectional ion flux studies using 36Cl
were then performed on confluent CCD cells grown on
0.33-cm2 Transwell filters. The internal diameter of the
filters used to grow the CCD cells and the small volume of apical
medium required made it impossible to collect the serial 50-µl
samples necessary for the isotopic flux measurements under
short-circuit current conditions. The Cl
flux studies
were therefore performed under the more physiological open-circuit
condition (8, 34). Continuous monitoring of RT and VT with the
Millicel electrical resistance clamp apparatus showed that
10
8 M dDAVP induced a slight decrease in
RT and a sustained increase in
VT that was more marked in CFTR(
/
) than in
CFTR(+/+) CCD cells (data not shown). As a consequence, the equivalent
short-circuit current (Ieq) calculated from
RT and VT was
significantly greater (P < 0.05) after dDAVP-treatment
in CFTR(
/
) CCD cells (44.5 ± 5.5 µA/cm2,
n = 19) than in CFTR(+/+) CCD cells (27.9 ± 3.1 µA/cm2, n = 21). The open-circuit
conditions therefore seemed to be suitable for ion flux studies. In
untreated CFTR(+/+) and CFTR(
/
) CCD cells,
36Cl
flux was slightly greater in the
apical-to-basal direction than the basal-to-apical direction (Fig.
8), indicating a net absorption of
Cl
. dDAVP doubled the basal-to-apical flux of
36Cl
in CFTR(+/+) CCD cells, but, in sharp
contrast, it had no impact on the basal-to-apical
36Cl
flux in CFTR(
/
) CCD cells (Fig. 8).
dDAVP also increased the apical-to-basal 36Cl
flux to a similar, significant extent in both CFTR(+/+) CCD cells (2.5-fold) and CFTR(
/
) CCD cells (2.9-fold) (Fig. 8). As a
consequence, the net absorption of Cl
measured under
open-circuit condition in CFTR(
/
) CCD cells was about twofold
greater than in CFTR(+/+) CCD cells.
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DISCUSSION |
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Using Isc experiments and
36Cl flux studies using primary cultures of
isolated CCDs microdissected from the kidneys of wild-type and
cftrm1unc mice, we have shown that
Cl
is secreted in response to cAMP stimulation and have
demonstrated the role of CFTR in this process.
Total blockade of ENaC by BAm and the use of Na+-free
solutions applied to the apical side of the cells grown on filters
almost completely inhibited the basal transport of Na+ but
did not impair the small rise in Isc
(corresponding, in this case, to a flow of negative charges from the
basal to the apical medium) induced by dDAVP or ISO in cultured
wild-type CCD cells. This small component of Isc
can therefore be attributed to Cl secretion, because it
occurs in the presence of BAm and the absence of Na+ and is
inhibited, at least to some extent, by agents known to block
Cl
channels. The fact that NPPB, the most potent, but
nonselective, Cl
channel blocker (35)
tested, partially inhibited both dDAVP- and ISO-stimulated
Isc in amiloride-treated CFTR(+/+) CCD cells provides a further indication that CFTR is implicated in this process.
Previous studies have shown that cAMP agonists stimulate Cl
secretion in amphibian A6 cells (4, 19,
34) and mammalian CCD and IMCD cells (8, 20) and
that the electrogenic anion secretion by cultured IMCD cells can be
inhibited by inhibitors of the CFTR Cl
channel (14,
33). The detection of CFTR mRNA and CFTR-like Cl
currents in several cultures of DCT, CCD, and IMCD
cells of different origins has also provided further indirect evidence
of the involvement of CFTR in the Cl
-secreting pathway
(7, 14, 17, 22). Furthermore, Morris et al.
(19) have shown that vasotocin, which stimulates
Na+ absorption and secretory Cl
currents in
amphibian A6 cells, concomitantly induced an apparent microtubule-dependent recruitment of cytoplasmic CFTR to the apical cell surface. In this study, we have shown that the component of
Cl
secretion elicited by dDAVP in mouse wild-type CCD
cells is partially inhibited by NPPB and, to a lesser extent, Glb, but
not by DIDS. This pharmacological inhibition profile closely resembles
previously reported observations that were attributed to CFTR, in
murine kidney cultured cells (14, 22). Thus the results
obtained in mouse CFTR(+/+) CCD cells in primary culture agree well
with those of similar studies of other cultured renal cells.
The implication of CFTR in the cAMP-stimulated Cl
secretory pathway in CCD was directly demonstrated by showing that
dDAVP and ISO failed to stimulate Isc in
BAm-treated CFTR (
/
) CCD cells and that the anion channel blockers
NPPB and Glb had no effects. Our results therefore demonstrate that the
Cl
diffusion pathway stimulated by cAMP agonists in the
apical membrane of cultured mouse CCD cells is formed by CFTR. The CFTR
protein is expressed in most, if not all, parts of the renal tubule,
including the distal collecting duct system. Results from RT-PCR
experiments confirmed that CCDs microdissected from wild-type mouse
kidneys express CFTR mRNA. However, the precise cellular
localization of the CFTR protein in the CCD (principal and/or
intercalated cells) has not yet been unambiguously identified. Not all
the anti-CFTR antibodies that we tested were suitable for performing a
proper immunofluorescence study to determine the precise localization of the protein in frozen mouse kidney sections and cultured mouse CCD
cells. CFTR mRNA has been found in both principal and
intercalated cells from immunodissected rabbit CCD cells, with the
highest level of expression found in the
-intercalated cells
(32). Letz and Korbmacher (17) also showed
that principal cells in culture coexpress CFTR and ENaC. Here, we have
shown that dDAVP stimulates a predominent component of
amiloride-sensitive Na+ absorption mediated by ENaC in both
CFTR(+/+) and CFTR(
/
) CCD cells, as well as a minor component of
Cl
secretion, which is inhibited by NPPB in CFTR(+/+) but
not CFTR(
/
) CCD cells. These results strongly suggest that CFTR is
coexpressed with ENaC in principal cells. The fact that ISO, which does
not stimulate Na+ transport, also increases the secretion
of Cl
in CFTR(+/+) cells but not in CFTR(
/
) CCD
cells, indicates that functional CFTR is also expressed in intercalated
cells. dDAVP plus ISO also has a greater impact on NPPB-sensitive
Isc than dDAVP alone, and this once again is
consistent with the presence of CFTR in both principal and intercalated cells.
Besides the fact that an electrogenic secretion of Cl can
be demonstrated by the Isc method (4, 8, 14, 20, and the present study), vasopressin (or vasotocin) has also been shown
preferentially to increase net Cl
absorption by cultured
A6 or CCD cells under more physiological, open-circuit conditions
(7, 20, 34). We also found that dDAVP stimulates the
basal-to-apical component of Cl
flux measured in
CFTR(+/+) cells but has no effect on CFTR(
/
) CCD cells. However,
dDAVP also produces a similar increase in the predominant
apical-to-basal component of Cl
flux in both CFTR(+/+)
and CFTR(
/
) cells. Because only Cl
secretion can be
detected under short-circuit conditions, the results obtained under the
more physiological, open-circuit condition indirectly suggest that the
apical-to-basal flux of Cl
stimulated by dDAVP occurs via
the paracellular pathway (25). All together, the net
cAMP-stimulated Cl
absorption observed in cultured cells
under open-circuit conditions (8, 20, 33, and the present study) is in
agreement with in vivo data demonstrating net Cl
absorption in isolated, microperfused CCD tubules (25).
Although the presence of apical Cl
conductance has been
documented in cultured CCD cells as well as in isolated, perfused CCD
tubules (24), the evidence that Cl
secretion
is part of Cl
transport across CCD in vivo is poorly
documented (27) and relies almost entirely on the fact
that microperfused CCD tubules dissected from rabbits fed a
K+-rich diet exhibit active Cl
secretion
(36).
It is now clear that CFTR, besides acting as a Cl
channel, can modulate the activity of several ion channels, including
ENaC (3, 30, 31). The question arises whether CFTR can
modulate Na+ absorption mediated by ENaC in principal CCD
cells. We found that the amiloride-sensitive component of
dDAVP-stimulated Isc, which reflects
ENaC-mediated Na+ absorption, although slightly higher, was
not significantly increased in CFTR(
/
) compared with CFTR(+/+) CCD
cells. Thus CFTR has no effect on the basal Na+ transport
and only slightly decreases the stimulatory effect of cAMP on
Na+ transport in CCD. In contrast to what is normally
observed in CCD, Boucher et al. (2) found that cAMP
inhibits rather than stimulates Na+ absorption in normal
nasal epithelial cells. These authors reported that basal
Na+ absorption is higher in airway cells from CF patients
than in normal cells and that it could be stimulated by cAMP. Enhanced basal Na+ absorption also occurs in the nasal epithelium
and colon from the cftrm1unc mice (10,
11). In sharp contrast with the above studies, Reddy et al.
(21) recently reported that ENaC activity is dependent on,
and increases with, CFTR in freshly isolated normal sweat glands and
that Na+ conductance is lower in CF sweat glands. The
possibility cannot be excluded that there may be different regulatory
pathways in different cell systems with differing vectorialized ion
transport properties such as CCD, where CFTR mediates Cl
secretion, and sweat glands, where CFTR mediates Cl
absorption. Hence, disrupting CFTR in the CCD seems to have a marginal
effect on Na+ absorption compared with that reported for
most CFTR target tissues. The cellular heterogeneity from intact CCDs
and primary cultured CCD cells may explain the lack of apparent
interaction between CFTR and ENaC, just because intercalated cells
(lacking ENaC) contain much more CFTR than principal cells and that,
under most circumstances, there will be no or very poor interactions
between ENaC and CFTR. Another possible explanation could be that the level of expression of CFTR in principal CCD cells remains
very low and is therefore insufficient to regulate the predominant, more highly expressed ENaC in these cells. This would
explain why the rate of Na+ absorption (which accounts for
>80% of the total Isc measured) stimulated by
dDAVP in CFTR(+/+) CCD cells does not change much when CFTR is
disrupted and why an increase in fractional Na+ excretion
can be demonstrated only under particular circumtances in CF mice
(15).
In conclusion, this study provides direct evidence that CFTR mediates
Cl secretion, a minor component of overall
Cl
transport, in primary-cultured mouse CCD cells and
suggests that the increase in NaCl absorption elicited by vasopressin
in vivo is not greatly different in CCD cells in which functional CFTR is lacking.
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ACKNOWLEDGEMENTS |
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We thank M. F. Bertrand (Centre de Développement des Techniques Avancées pour l'Expérimentation Animale, Orleans, France) and the Association Française de Lutte contre la Mucoviscidose, who kindly provided us the cftrm1Unc mice. We thank M. Ghosh for editing assistance.
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FOOTNOTES |
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This study was supported by INSERM and in part by a grant from the "Mucoviscidose:ABCF Proteins" and the Association pour l'aide à la recherche contre la mucoviscidose et l'assistance aux malades.
Present address of J.-P. Duong Van Huyen: Service d'Anatomie Pathologique, Hôpital Georges Pompidou, 75015 Paris, France.
Address for reprint requests and other correspondence: A. Vandewalle, INSERM U478, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris, Cédex 18, France (E-mail: vandewal{at}bichat.inserm.fr).
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 16 February 2001; accepted in final form 27 April 2001.
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