In vivo activation of CFTR-dependent chloride transport in
murine airway epithelium by CNP
Thomas J.
Kelley1,
Calvin U.
Cotton1, and
Mitchell L.
Drumm1,2
Departments of 1 Pediatrics and
2 Genetics and Center for Human
Genetics, Case Western Reserve University, Cleveland, Ohio
44106-4948
 |
ABSTRACT |
Inhibitors of
guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited
phosphodiesterases stimulate
Cl
transport across the
nasal epithelia of cystic fibrosis mice carrying the
F508 mutation
[cystic fibrosis transmembrane conductance regulator (CFTR)
(
F/
F)], suggesting a role for cGMP in regulation of
epithelial ion transport. Here we show that activation of
membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP)
stimulates hyperpolarization of nasal epithelium in both wild-type and
F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(
/
)]. With the use of a nasal
transepithelial potential difference (TEPD) assay, CNP was found to
hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying
wild-type CFTR. This value is consistent with that obtained with
8-bromoguanosine 3',5'-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP
demonstrated a synergistic ability to induce
Cl
secretion across the
nasal epithelium of CFTR(
F/
F) mice. No effect on TEPD was seen
with this combination when used on CFTR(
/
) mice, implying
that the CNP-induced change in TEPD in CFTR(
F/
F) mice is CFTR
dependent.
guanylate cyclase;
F508 cystic fibrosis transmembrane
conductance regulator; pharmacological activation; C-type natriuretic
peptide
 |
INTRODUCTION |
NATRIURETIC PEPTIDES are known regulators of fluid and
ion transport in several systems (reviewed in Ref. 25) and have been shown to regulate Cl
transport in shark rectal gland cells (28) and in porcine colon via a
guanosine 3',5'-cyclic monophosphate (cGMP)-dependent
mechanism (1). Similarly, both heat-stable enterotoxin (Sta) and
guanylin, peptides structurally and functionally related to the
natriuretic peptides, have been shown to stimulate
Cl
transport in the human
colonic cell lines T84 and Caco-2 (3, 5) through a cystic fibrosis
transmembrane conductance regulator (CFTR)-dependent pathway.
The regulatory actions of the natriuretic peptides are thought to be
mediated predominantly through cGMP. The natriuretic peptides bind to
and activate particulate, transmembrane receptors that contain
intracellular guanylate cyclase (GC) domains. Each of the natriuretic
peptides, atrial (ANP), brain (BNP), and C-type (CNP), have varied
affinities for specific receptors. ANP and BNP both bind to GC-A, with
ANP having the greater affinity, and CNP binds to the GC-B receptor
(reviewed in Ref. 32).
Although there is an apparent specificity of function for ANP and BNP
in cardiac tissue and in the pulmonary vascular system compared with
CNP (9, 20, 21, 29), CNP binding and function predominates in airway
epithelial cells (6, 7, 30). Specific binding of CNP to human airway
epithelial cells has been demonstrated by the functional ability of CNP
to elevate cGMP levels in these cells. ANP had no significant effect on
cGMP levels, although sodium nitroprusside (SNP), an activator of
soluble GCs, did elevate cGMP concentrations in this system.
Additionally, CNP was shown to increase ciliary beat frequency in
primary airway epithelial cells, implying a role for CNP in the
regulation of mucociliary clearance (6) and raising the possibility
that CNP may play a role in the regulation of other nonrespiratory
functions in the airway.
We have previously shown that cGMP-inhibited phosphodiesterases
(cGI-PDEs) represent a key step in the regulatory pathway of the CFTR
in human airway epithelial cells (12). Inhibition of cGI-PDEs with the
cardiotonic drug milrinone stimulates CFTR activity in cells that
express either wild-type or
F508 forms of CFTR (11, 14). The
specificity of cGI-PDEs compared with other PDE classes in the
regulation of CFTR activity clearly implies an important role for cGMP
in this process. To establish this regulatory role for cGMP, we have
also shown that the addition CNP can mimic the effects of milrinone and
can lead to the stimulation of CFTR activity in both Calu-3 and CF-T43
cells (13). These results are completely consistent with
the previously mentioned reports that demonstrate CFTR activity in
colonic cells in response to guanylin and Sta (3, 5).
Although we have shown that CNP is capable of regulating
Cl
secretion in cultured
airway epithelial cells, the possible paracrine or autocrine regulation
of CFTR activity by natriuretic peptides has not been established. The
first step in this process is to demonstrate stimulation of CFTR in
intact tissue by CNP. We have chosen the mouse as a model in which to
study this system because the CNP gene is conserved between mice and
humans (22, 23) and because CFTR null
[CFTR(
/
)] mice are available to serve as
controls for CNP-mediated CFTR activation (2, 8, 17, 26). In this
paper, in vivo mouse nasal transepithelial potential difference (TEPD)
measurements as well as in vitro mouse tracheal TEPD measurements were
made in response to apically applied CNP and other GC agonists to
demonstrate their ability to regulate Cl
transport across murine
respiratory epithelium.
 |
MATERIALS AND METHODS |
Peptides.
Both CNP and vasonatrin were purchased from Bachem Bioscience.
Measurement of mouse nasal TEPD values.
Mouse nasal TEPD was measured by the protocol of Grubb et al. (8).
Briefly, mice were anesthetized with 200 µl/20 g body wt of 0.4 mg
acepromazine, 11 mg ketamine, and 2 mg xylazine/ml phosphate-buffered
saline. PE-10 tubing drawn out to approximately one-half of its
original diameter was inserted 2-3 mm into the nostril of the
mouse. Solutions were perfused at room temperature with the use of a
Razel A-99 (Razel Scientific Instruments, Stamford, CT) syringe pump at
a rate of ~7 µl/min. Each syringe was bridged to a Calomel
electrode through a 4% agar bridge made up in either Cl
-replete or
Cl
-free Ringer solution. An
~10-mV junction potential in the negative direction was instantly
seen upon switching to
Cl
-free solutions. This
junctional potential is not shown in Figs. 1-9 and is
not corrected for in the values given. A series of valves was used to
change solutions, with a delay time of ~45 s between solution change
and solution contact with the nasal epithelia. Ringer solutions
consisted of Cl
-replete
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-buffered Ringer solution [HBR; containing (in
mM) 10 HEPES (pH 7.4), 138 NaCl, 5 KCl, 2.5 Na2HPO4,
1.8 CaCl2, and 1.0 MgSO4] and
Cl
-free HBR
[containing (in mM) 10 HEPES (pH 7.4), 138 sodium gluconate, 5 potassium gluconate, 2.5 Na2HPO4,
3.6 hemicalcium gluconate, and 1.0 MgSO4; all chemicals were from
Sigma Chemical, St. Louis, MO]. Some mice were studied on
multiple occasions. Mice that were retested were given at least 48 h
between assays.
Measurement of mouse tracheal potential difference values.
Excised tracheae were mounted on hold pipettes (30-ml
micropipettes; Drummond Scientific) inserted into each end of
the trachea, and the luminal and bath compartments were perfused
independently. All experiments were performed at 37°C by placing
all solutions and the trachea-mounting chamber in an Isolette infant
incubator (Narco, Hatboro, PA). HBR was perfused to both luminal and
basolateral sides by gravity. Luminal perfusion rates were between 3 and 5 ml/min. TEPD was measured via 4% agar bridges in HBR placed on both luminal and basal sides and was connected through calomel electrodes to a DVC 1000 voltage-current clamp (WPI). Data were collected on a MacLab/4e from Advanced Instruments.
cGMP measurements.
Mouse tracheal tissue was excised and cut into three equal sections for
each experiment. The epithelial layer was not isolated for these
experiments, so various cell types were present, including neuronal
cells. The tissue was incubated for 5 min at 37°C in physiological
salt solution buffered by 10 mM tris(hydroxymethyl)aminomethane (Tris;
pH 7.5) containing either 1 µM CNP or 300 µM SNP or in the absence
of any GC agonist as a control. Samples were then placed in 0.5 ml of
10 mM Tris (pH 7.5) and 100 µM EDTA at 90°C for 10 min. Samples
were then placed on ice, homogenized, and spun down in a microfuge at
14,000 revolutions/min for 5 min, and the supernatant was collected
for analysis. Measurements of cGMP levels were performed
using an enzyme-linked immunoassay kit, according to the
manufacturer's instructions (Cayman Chemical, Ann Arbor, MI).
Mice.
Mice were genotyped from tail clip DNA.
F508 mice were a generous
gift from Kirk Thomas of the University of Utah School of Medicine and
were genotyped by the procedures described previously (33).
CFTR(
/
) mice (27) were obtained from Jackson Laboratories and were genotyped as described by Koller et al. (16). To increase survival of cystic fibrosis animals, mice were fed a liquid diet as
described by Eckman et al. (4). Mice were cared for in accordance with
Case Western Reserve University guidelines.
 |
RESULTS |
Effects of cGMP agonists on mouse nasal TEPD.
Three GC agonists and a cGMP analog, 8-bromoguanosine
3',5'-cyclic monophosphate (8-BrcGMP), were tested by nasal
TEPD assay for their ability to stimulate
Cl
secretion in the nasal
epithelia of mice when perfused onto murine nasal epithelia in
Cl
-free HBR (Fig.
1). An agonist of cytosolic GCs, SNP (300 µM), had very little effect on TEPD values, hyperpolarizing lumen
negative TEPD only 2.4 ± 0.9 mV (n = 7). In contrast, 1 µM CNP, which activates GC-B, a membrane-bound
GC, stimulates a change in TEPD (
TEPD) of 6.1 ± 0.6 mV
(n = 8), representing lumen negative
hyperpolarization. Similarly, the GC-B-specific peptide vasonatrin (1 µM), a chimeric peptide that contains the bioactive loop region of
CNP, and the carboxy-terminus of ANP hyperpolarized lumen negative TEPD
6.5 ± 1.1 mV (n = 4). Consistent
with these effects being mediated by increased cGMP production,
addition of the analog 8-BrcGMP (100 µM) resulted in a
TEPD of
6.2 ± 0.9 mV (n = 3).

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Fig. 1.
Changes in nasal transepithelial potential difference ( TEPD) in
response to guanylate cyclase agonists and 8-bromoguanosine
3',5'-cyclic monophosphate (8-BrcGMP). Plots show the
TEPD in individual cystic fibrosis transmembrane conductance
regulator (CFTR) (+/+), CFTR(+/ ), and CFTR(+/ F) mice to 1 µM C-type natriuretic peptide (CNP)
(A), 1 µM vasonatrin
(B), 100 µM 8-BrcGMP
(C), and 300 µM sodium
nitroprusside (SNP) (D). All traces
are performed in the presence of 100 µM amiloride in
Cl -free HEPES-buffered
Ringer solution (HBR). Before refers to the TEPD 30 s before drug
addition. After shows the maximal TEPD obtained within 2.5 min after
drug addition. Average TEPDs of each experiment are shown by the black
bars, and average TEPDs ± SE are given at
bottom.
* P < 0.05;
** P < 0.005;
*** P < 0.0001. Paired
t-tests were performed to determine
significance.
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Pharmacological characterization of CNP-mediated
Cl
secretion in mouse nasal
epithelia.
Previously, we reported that CNP stimulated CFTR-mediated
Cl
current in Calu-3 cells
as determined by whole cell patch-clamp recordings (13). To determine
if CFTR has a role in CNP-stimulated Cl
secretion in vivo, the
Cl
channel blockers
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and
diphenylamine-2-carboxylate (DPC) were used to pharmacologically
characterize responses to CNP (18, 31). DPC (1 mM) reduced
CNP-stimulated changes in mouse nasal TEPD ~70%, whereas 500 µM
DIDS did not have a significant effect (Fig. 2). This inhibitor sensitivity profile is
consistent with CFTR activation. For comparison, forskolin-stimulated
nasal TEPD changes were also tested in the presence of DPC and DIDS.
Forskolin-stimulated
TEPDs were reduced ~55% by DPC and were
unaffected by the presence of DIDS (Fig.
2B).

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Fig. 2.
Effect of Cl channel
blockers on CNP-mediated TEPDs. A:
averaged traces of nasal TEPD assays performed on CFTR(+/+),
CFTR(+/ ), and CFTR(+/ F) mice not treated with channel
blockers or in the presence of either 500 µM DIDS or 1 mM
diphenylamine-2-carboxylate (DPC). Values for changes in TEPD were
taken at 15-s intervals and were plotted as TEPD. Addition of
amiloride and CNP in the assay is indicated by the black bars. Zero
TEPD and time 0 correspond to the point at which
1 µM CNP was added. All traces were performed in the presence of 100 µM amiloride in Cl -free
HBR. Positive TEPD values indicate a hyperpolarization of lumen
negative potential difference. Error bars represent SE.
B: bar graph comparing the effects of
DIDS and DPC treatment on TEPD values stimulated by either 1 µM
CNP or 10 µM forskolin (Forsk) after 2 min. Error bars represent SE,
and the number of experiments (n) is
shown in parentheses. Comparisons were made with each condition to
Forsk. Significance was determined by Duncan's multiple-range test
with = 0.05 with a Bonferroni correction for multiple comparisons.
Significance corresponds to P < 0.01. * P < 0.001;
** P < 0.0001.
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To more directly test for CFTR activity in response to CNP,
CFTR(
/
) mice were compared with wild-type mice in their
ability to respond to CNP. CNP (1 µM), in
Cl
-free HBR and in the
presence of amiloride, was perfused into the nasal cavity of
CFTR(
/
) mice as described for the wild-type mice. The
CFTR(
/
) mice failed to show any hyperpolarization of
lumen negative potential in response to either
Cl
-free HBR alone or with
the agonist CNP (Fig. 3). These data, coupled with the inhibitor profile of CNP-induced changes in TEPDs, are
strong evidence that CNP stimulates epithelial
Cl
transport by regulating
CFTR activity.

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Fig. 3.
CNP increases TEPD in CFTR(+/+) and CFTR(+/-) mice. Averaged traces of
nasal TEPD assays performed on CFTR(+/+) and CFTR(+/-) mice
(n = 3) and on CFTR( / )
mice (n = 4). Values were taken at
15-s intervals and were plotted as TEPD. The addition of amiloride
and CNP in the assay is indicated by the black bars. Zero TEPD and
time 0 correspond to the point at which 1 µM CNP was added. All traces are performed in the presence of 100 µM amiloride in Cl -free
HBR. Positive TEPD values indicate a hyperpolarization of lumen
negative potential difference. Error bars represent SE.
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To show that CNP was acting through its receptor GC-B, two inhibitors
of GC activity were used (Fig. 4). First,
perfusion with the compound LY-83583 (50 µM), which directly inhibits
GC activity (15, 19), completely inhibited any response to CNP. Second,
perfusion with 250 nM phorbol 12,13-dibutyrate (PDBu) inhibited
CNP-mediated changes in TEPD by ~90%, consistent with reports that
phorbol esters cause protein kinase (PK) C-dependent desensitization of
natriuretic peptide receptors (24). For comparison, PDBu had no effect
on forskolin-stimulated changes in TEPD, indicating that inhibition of
the CNP effect was not due to PDBu interfering the ability of CFTR to
activate.

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Fig. 4.
CNP-mediated TEPDs in the presence of guanylate cyclase antagonists.
A: averaged traces of nasal TEPD
assays performed on CFTR(+/+), CFTR(+/ ), and CFTR(+/ F) mice
not treated with guanylate cyclase inhibitors or in the presence of
either 250 nM phorbol 12,13-dibutyrate (PDBu) or 50 µM LY-83583.
Values were taken at 15-s intervals and were plotted as TEPD.
Addition of 100 µM amiloride and 1 µM CNP in the assay is indicated
by the black bars. Zero TEPD and
time 0 correspond to the point at which CNP
was added. Positive TEPD values indicate a hyperpolarization of
lumen negative potential difference. Error bars represent SE.
B: bar graph comparing the effects of
PDBu treatment on TEPD values stimulated by either 1 µM CNP or 10 µM Forsk. Error bars represent SE, and
n is shown in parentheses. Statistical
analysis is a comparison of the responses to the indicated agents with
the responses to Forsk. Duncan's multiple range test with a Bonferroni
correction for multiple comparisons was used. Significance at = 0.05 corresponds to P < 0.013. * P < 0.0001.
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Stimulation of Cl
secretion in
mouse trachea by CNP.
To determine if CNP acts on epithelia in other portions of the airway,
the effects of CNP on Cl
transport were tested by measuring TEPD values in excised mouse trachea. We first established that the CNP receptor was present and
functional in tracheal tissue by measuring cGMP production in response
to 1 µM CNP and 300 µM SNP (Fig.
5). Compared with tissues not incubated
with GC agonists, SNP increased cGMP levels ~2-fold, whereas CNP
increased cGMP levels ~50-fold.

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Fig. 5.
Elevation of cGMP values in response to either 300 µM SNP or 1 µM
CNP in trachea of CFTR(+/+) or CFTR(+/ ) mice. Determinations of
cGMP content were performed as described in MATERIALS
AND METHODS. Error bars represent SE, and
n is shown in parentheses.
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Whereas the nasal TEPD assays were performed by perfusing
Cl
-free Ringer solution
onto the surface of murine nasal epithelium to increase the
electrochemical driving force for
Cl
secretion, the tracheal
TEPD assays were performed with
Cl
-replete Ringer solution
on both the basolateral and luminal sides. Under these conditions, CNP
alone was not sufficient as a stimulus to generate hyperpolarization in
tracheal epithelium and resulted in further depolarization of TEPD.
Forskolin alone did generate a detectable hyperpolarization of TEPD in
the trachea. However, a combination of forskolin plus CNP stimulated a
twofold greater
TEPD than forskolin alone (Fig.
6). This synergistic relationship between
forskolin and CNP suggests an enhancement of the adenosine 3',5'-cyclic monophosphate-PKA pathway rather than additive
effects of two separate pathways.

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Fig. 6.
In vitro TEPD changes of a wild-type mouse trachea in response to Forsk
(Fsk) and CNP. A: raw trace of
tracheal TEPD assay showing responses to luminal applications of 100 µM amiloride (A), 10 µM Fsk, and 1 µM CNP. HBR represents
perfusion with Cl -replete
HEPES-buffered Ringer solution. Reduction (depolarization) in TEPD
caused by amiloride is partially restored by the addition of Fsk. After
washout with HBR, the TEPD returns (hyperpolarizes) to baseline.
Treatment with amiloride again depolarizes the TEPD, and CNP has no
appreciable effect. After a second washout and depolarization with
amiloride, Fsk is added a second time. Basal TEPD values for these
experiments were 2.8 ± 0.3 mV
(n = 4).
B: bar graph showing TEPD in
response to the first addition of Fsk alone (Fsk1;
n = 3), CNP alone (CNP;
n = 4), Fsk plus CNP (Fsk + CNP;
n = 3), and a second addition of Fsk
(Fsk2; n = 3). Significance is
measured against responses to Fsk. Positive TEPD values indicate a
hyperpolarization of lumen negative potential difference. Error bars
represent SE. Statistical analysis is a comparison of the responses to
the indicated agents with the responses to Fsk. Duncan's multiple
range test with a Bonferroni correction for multiple comparisons was
used. Significance at = 0.05 corresponds to
P < 0.017. * P < 0.0005.
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In vivo activation of
F508 CFTR by forskolin and
CNP.
We have previously shown that
Cl
transport can be
stimulated in the nasal epithelium of
F508/
F508 mice with the
combination of forskolin and the cGI-PDE inhibitor milrinone
but not with either compound alone (14). Activation of
Cl
secretion was not
observed when nasal epithelium of CFTR(
/
) mice was
exposed to these compounds. Our hypothesis from these data is that both
CNP and milrinone are acting to stimulate CFTR activity by inhibition
of cGI-PDEs. If true, the combination of forskolin and CNP, like
forskolin and milrinone, should stimulate Cl
secretion in
F508/
F508 mice.
Mouse nasal TEPD assays with
F508/
F508 mice showed that neither
forskolin nor CNP alone stimulates a hyperpolarization of TEPD, as we
had found with milrinone alone. Consistent with our hypothesis, after a
2-min exposure to forskolin plus CNP, TEPD was hyperpolarized 3.4 ± 1.0 mV (n = 8) compared with a
depolarization of 2.1 ± 0.6 mV (n = 3) induced by forskolin or a 2.1 ± 0.9 mV (n = 3) depolarization induced by CNP
alone (Fig. 7). This 3.4 ± 1.0-mV
hyperpolarization of TEPD in CFTR(
F/
F) mice is ~35% of the
amount of activation achieved with the combination of forskolin and CNP
in CFTR(+/
F) mice (9.7 ± 1.3 mV;
n = 3), whereas forskolin plus CNP
induced a 2.3 ± 0.8-mV (n = 4)
depolarization of TEPD in CFTR(
/
), implicating a role for
CFTR in the response to this combination of compounds. One differential
response of CFTR(
/
) mice and CFTR(
F/
F) mice is
illustrated in Fig. 8. These data show that
CFTR is involved in the mechanism of CNP-induced
Cl
secretion in airway
epithelia of mice.

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Fig. 7.
Stimulation of CFTR-dependent TEPDs in mouse nasal epithelia.
A: averaged traces of nasal TEPD
assays performed on CFTR( F/ F) mice treated with either 10 µM
Forsk (n = 3), 1 µM CNP
(n = 3), or 10 µM Forsk plus 1 µM
CNP (n = 8). Zero TEPD and
time 0 correspond to the point at which
agonists were added. All traces were performed in the presence of 100 µM amiloride in Cl -free
HBR. Positive TEPD values indicate a hyperpolarization of lumen
negative potential difference. Error bars represent SE.
B-D:
raw traces of Forsk alone (B), CNP
alone (C), and Forsk plus CNP
(D). Agonists were added at the
2-min time point (arrows).
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Fig. 8.
Averaged traces of nasal TEPD assays performed on CFTR
( F/ F) mice (n = 8) or
CFTR( / ) mice (n = 4)
treated with 10 µM Forsk plus 1 µM CNP. Values were taken at 15-s
intervals and were plotted as a TEPD. Zero TEPD and
time 0 correspond to the point at which
agonists were added. All traces were performed in the presence of 100 µM amiloride in Cl -free
HBR. Positive TEPD values indicate a hyperpolarization of lumen
negative potential difference. Error bars represent SE.
B and
C: raw traces of CFTR( F/ F)
mice and CFTR( / ) mice treated with Forsk plus CNP,
respectively. Agonists were added at the 2-min time point.
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 |
DISCUSSION |
We have previously shown that cGI-PDEs are an important part of the
CFTR regulatory pathway (11, 12, 14). The involvement of this PDE class
suggests an important role for cGMP in the normal regulation of
Cl
transport across
CFTR-expressing epithelium. This notion is supported by other reports
that show CFTR activation in human colonic cells in response to both
Sta and guanylin, both of which are peptides that activate
membrane-bound GCs (3, 5). A similar peptide, CNP, has been shown to
stimulate cGMP production in primary cultures of human airway
epithelial cells (7). We have demonstrated in cultured lung epithelial
cells (Calu-3) that CNP is capable of stimulating CFTR activity as
shown by whole cell patch-clamping experiments (13). This stimulation
of activity was mediated by cGMP but via a PKA-dependent pathway,
consistent with our previous results from the cGI-PDE inhibitor
milrinone.
Natriuretic peptides have been shown to regulate fluid and electrolyte
transport in several systems (1, 19, 25, 28). The ability of CNP to
stimulate cGMP production in human airway epithelial cells suggests a
role for CNP in the regulation of airway electrolyte transport. Our
data show that CNP can stimulate Cl
secretion when applied
to the apical membrane of mouse nasal epithelia in vivo. This
stimulated Cl
transport has
the pharmacological characteristics of CFTR and is not present when CNP
is applied to the nasal epithelium of CFTR(
/
) mice.
It has also been reported, however, that CNP has no effect on
Cl
transport when applied
to cultured monolayers of ciliated human nasal epithelial cells
obtained from nasal scrapings (6). A possible reason for this
discrepancy is that culture conditions for primary cells are not
optimal for facilitating responses to CNP, thus reducing
reproducibility and magnitude of CNP-mediated stimulation. Thus our in
vivo measurements of nasal TEPD and our in vitro studies with freshly
excised trachea may provide a more appropriate model than studies with
cultured cells. A methodological difference between the studies is that
our nasal TEPD measurements were made with the luminal side of the
epithelium perfused with Cl
-free HBR, whereas the
short-circuit current measurements made in the previous report were in
symmetrical Cl
-replete
Krebs-buffered Ringer (KBR). The short-circuit data are consistent with
our findings in freshly excised trachea in KBR (Fig. 6) in which we
were unable to see a response with CNP in the presence of amiloride. We
could demonstrate, however, a synergistic increase in
forskolin-mediated hyperpolarization of TEPD by CNP. Another possible
difference between systems may lie in the fact that CNP and its
receptor GC-B have been identified in brain tissue and nerve fibers
(reviewed in Ref. 10). It is possible that in our in vivo system CNP is
interacting with nerve fibers within the nasal passage and stimulating
the release of other factors that may be initiating CFTR activation in
the epithelium. This pathway would not be available in cultured
monolayers of harvested primary epithelial cells. We think this is
unlikely to explain the difference seen here, as our previous study in
Calu-3 and CF-T43 cells showed that cultured epithelial cells exposed
to CNP are sufficient to stimulate CFTR-dependent
Cl
transport (13).
The demonstrated ability of natriuretic peptides to generically
regulate salt and fluid transport in several systems opens the
possibility that an in vivo role of CNP may be to regulate Cl
transport in the airways
through CFTR. The similarity of effects that both cGI-PDE inhibitors
and the GC agonist CNP have on CFTR activity implies a regulatory role
for cGMP in ion transport regulation across airway epithelial cells.
The specificity of CNP-mediated regulation of CFTR activity extends to
its ability to stimulate Cl
transport across nasal epithelia of CFTR(
F/
F) mice when used in
combination with the adenylate cyclase agonist forskolin but has
no effect on Cl
secretion in CFTR(
/
) mice. These results are
consistent with our previous data demonstrating that the combination of
forskolin and the cGI-PDE inhibitor milrinone stimulates
F508 CFTR
activity in the nasal epithelia of
F/
F mice and support the
hypothesis that milrinone is acting via a cGMP-dependent pathway (Fig.
9). The ability to stimulate
Cl
secretion in the nasal
epithelium of
F/
F mice in an in vivo system demonstrates the
therapeutic potential of CNP as a pharmacological agent and the
possible importance of CNP as an in vivo regulator of CFTR function.

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Fig. 9.
Schematic diagram showing the proposed mechanism of CNP-mediated CFTR
activation. PKA, protein kinase A; PDE, phosphodiesterase; GC,
guanylate cyclase. ( ) Inhibitory effect.
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 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
HL-50160 and DK-51878 and by grants from the Cystic Fibrosis Foundation.
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FOOTNOTES |
Address for reprint requests: M. L. Drumm, Dept. of Pediatrics, Case
Western Reserve Univ., 8th floor BRB, 10900 Euclid Ave., Cleveland, OH
44106-4948.
Received 21 February 1997; accepted in final form 7 August 1997.
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REFERENCES |
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