Am J Physiol Lung Cell Mol Physiol
281: L1173–L1179, 2001.
Examining basal chloride transport using the nasal
potential difference response in a murine model
KRISTINE G. BRADY,1 THOMAS J. KELLEY,2 AND MITCHELL L. DRUMM1,2
Center for Human Genetics, Department of Genetics, and 2Department
of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106-4948
1
Received 5 February 2001; accepted in final form 11 July 2001
(CF) is an autosomal recessive disorder
caused by mutations in the gene encoding the cystic
fibrosis transmembrane conductance regulator (CFTR;
see Refs. 17, 25, 26). CFTR can function as a cAMPregulated chloride channel (2, 3) and also appears to
affect the function of other ion channels, including the
epithelial sodium channel (12, 33), the outward rectifying chloride channel (ORCC; see Refs. 7 and 27), and
the renal inward rectifying potassium channel (22).
Most of the characterization of CFTR as an ion channel
or regulator of other channels has been restricted to
cell lines and heterologous expression systems in part
because of the limitations of in vivo assays. An exception is utilization of the transepithelial potential dif-
ference (TEPD) measurement in vivo as a means to
monitor electrophysiological changes across an intact
tissue.
The nasal TEPD measurement was originally described (18, 19) as a means to discriminate between CF
and non-CF individuals based on bioelectrical potential
difference (PD) properties that differ between the two
groups. With the use of this assay, it was observed that
the unstimulated basal PD measurement is hyperpolarized to a greater degree in CF compared with
non-CF tissue. This difference was shown to be predominantly the result of amiloride-sensitive sodium
absorption. In the presence of amiloride, the PD can be
hyperpolarized by imposing a chloride gradient on the
epithelium such that the luminal chloride concentration is low relative to the serosal fluid. The PD can be
further hyperpolarized by stimulation with agents that
raise cAMP levels. Both of these hyperpolarizing responses are absent or dramatically reduced in CF (20),
indicating that they are CFTR dependent at some
level.
It has been inferred from in vitro experiments that
the chloride permeability increase that occurs in response to increased cAMP levels is the result of activation of CFTR channels (6, 24). However, the mechanism responsible for the hyperpolarization in response
to a chloride gradient is more questionable. A reasonable hypothesis is that the low chloride response reflects the basal level of CFTR activity. Unfortunately,
genetic and nongenetic heterogeneity in humans
makes characterization of factors involved in the electrophysiological gradients measured by the PD difficult. However, with slight modification, this assay can
be applied to the mouse (8–10, 14–16, 23, 29, 30, 32) in
which both types of heterogeneity can be minimized.
We have found that the assay is very reproducible
when animals are maintained in a consistent manner
and matched for age and gender when assayed (unpublished observations). Our aim in using the PD assay
with a mouse model is to investigate the mechanisms
responsible for the low chloride response. Because this
response is absent in patients with CF, understanding
its physiological basis may aid in a greater understand-
Address for reprint requests and other correspondence: M. L.
Drumm, Dept. of Pediatrics, Case Western Reserve Univ., 8th floor
BRB, 10900 Euclid Ave., Cleveland, OH 44106-4948 (E-mail:
[email protected]).
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.
cystic fibrosis; cystic fibrosis transmembrane conductance
regulator; ion channel inhibitors; protein kinase A
CYSTIC FIBROSIS
http://www.ajplung.org
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society
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Brady, Kristine G., Thomas J. Kelley, and Mitchell L.
Drumm. Examining basal chloride transport using the nasal
potential difference response in a murine model. Am J
Physiol Lung Cell Mol Physiol 281: L1173–L1179, 2001.—
Epithelia of humans and mice with cystic fibrosis are unable
to secrete chloride in response to a chloride gradient or to
cAMP-elevating agents. Bioelectrical properties measured
using the nasal transepithelial potential difference (TEPD)
assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride
transport defects. Although the response to forskolin is CFTR
mediated, the mechanisms responsible for the response to a
chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to
low chloride and forskolin in vivo. Both responses show little
correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the
presence of several chloride channel inhibitors, including
DIDS, diphenylamine-2-carboxylate, glibenclamide, and
5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein
kinase A inhibitor, the Rp diastereomer of adenosine 3⬘,5⬘cyclic monophosphothioate (Rp-cAMPS). The responses to
low chloride and forskolin demonstrate significantly different
pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the
low chloride response.
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DISTINCT MECHANISMS OF EPITHELIAL CHLORIDE SECRETION
ing of the pathophysiology in CF. If channels other
than CFTR are involved, their identification could potentially provide additional therapeutic targets.
MATERIALS AND METHODS
AJP-Lung Cell Mol Physiol • VOL
RESULTS
Effect of CFTR(⌬F508/⌬F508) mutation on the nasal
TEPD. The TEPD measurement predominantly reflects the level of sodium and chloride transport across
the airway epithelium in vivo. For this work, the TEPD
assay was separated into two separate traces to increase mouse survival. First, a baseline TEPD was
measured while the epithelium was perfused with an
isotonic Ringer solution until a stable value was determined. A second trace was made up of three distinct
phases. Initially, amiloride-sensitive sodium absorption was inhibited by perfusion with Ringer solution
containing amiloride. Once a steady-state value was
reached, a chloride gradient was created across the
epithelium by perfusion with chloride-free Ringer solution in which chloride ions were replaced with impermeant gluconate ions to stimulate chloride secretion into the lumen. Once a new steady state was
reached and the change in potential difference
(⌬TEPD) was determined, forskolin was added to the
chloride-free Ringer solution to stimulate adenylate
cyclase and raise cAMP. The response to cAMP was
measured again until a steady state was reached, and
the ⌬TEPD was determined again.
CF mouse models, including the Cftr ⌬F508 mice,
exhibited consistent ion transport characteristics similar to those seen in human CF patients (11, 31). The
differences between Cftr(⌬F508/⌬F508) mice and their
wild-type siblings are shown in Fig. 1 and are summarized in Table 1. The average baseline TEPD in isotonic
Ringer solution of CF mice was significantly more
negative than that of wild-type siblings because of the
increased sodium absorption seen in CF mice (non-CF:
⫺11.1 ⫾ 0.4 mV and CF: ⫺32.7 ⫾ 1.2 mV, P ⬍ 0.0001).
The addition of amiloride to the Ringer solution inhibited epithelial sodium transport, causing a depolarization in the PD in both CF and wild-type mice. However,
the average amiloride baseline measured in CF mice
was still significantly more negative than that of wildtype siblings (non-CF: ⫺3.4 ⫾ 0.3 mV and CF: ⫺10.6 ⫾
0.5 mV, P ⬍ 0.0001).
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Animals. Female mice of the A/J, C57BL/6J, DBA/2J,
BALB/cJ, C3H/HeJ, and 129/SvEms-⫹Ter?/J strains were
obtained from Jackson Laboratory (Bar Harbor, ME). Mice
with the Cftr mutation ⌬F508 on a mixed 129/SvEv and
C57BL/6J background (37) were a generous gift from Dr.
Kirk Thomas at the University of Utah School of Medicine.
As noted, in some experiments, mice used had the ⌬F508
mutation backcrossed onto the C57BL/6J background. Experiments were performed when the mice were between 12
and 31 wk of age. The project was approved by the Case
Western Reserve University Institutional Animal Care and
Use Committee.
Nasal PD measurements. The PD across the nasal epithelium of the mice was measured according to procedures
previously described elsewhere (11, 16), with some modifications. Briefly, mice were anesthetized with 11 mg/ml ketamine, 2 mg/ml xylazine, and 0.4 mg/ml acepromazine in
PBS. Animals were dosed intramuscularly with ⬃0.003 ml/g
mouse weight for baseline measurements and 0.0075 ml/g for
the assay in the presence of pharmacological agents requiring longer periods of anesthesia. A PE-10 tube stretched to
one-half the original diameter was inserted 2 mm into the
mouse nostril and placed against the septum. With the use of
a syringe pump (model A-99; Razel Scientific Instruments)
and 3-ml syringes, Ringer solution warmed to 37°C was
perfused into the nostril at a rate of ⬃7 ␮l/min. Valves
controlled which solution entered the nostril, with a 30-s
delay until the new solution reached the nasal epithelium.
Readings were taken until a steady-state value was reached
before perfusing the nasal epithelium with a new solution.
Filter paper wicks were used to absorb excess liquid from the
mouth and opposite nostril of the mouse being tested.
Bridges (4% agar) connected the tubing to calomel electrodes,
and each bridge was made in the same solution as the
perfusate it measured (HEPES-buffered Ringer or chloridefree HEPES-buffered Ringer). A needle containing 4% agar
in HEPES-buffered Ringer was placed subcutaneously in the
mouse’s back and connected to another calomel electrode to
serve as a reference. The measuring and reference calomel
electrodes were situated in a KCl bath in which one end of the
agar bridge was also immersed. The other end of the measuring electrode’s bridge was in contact with the perfusate,
⬃20 cm from the mouse nose. The transepithelial difference
was measured with a voltmeter (model ISO-DAM-D; World
Precision Instruments), and the signal was recorded on a
chart recorder (model BD112; Kipp and Zonen). Solution
resistance from the measuring electrode’s agar bridge to the
mouse nose was calculated to be ⬃1.2 ⫻ 106 ⍀, whereas input
resistance of the amplifier was ⬎1012 ⍀. The PD measurements were not corrected for junction potentials, and changes
in PD values were calculated by subtracting the final steadystate value from the first value taken after switching solutions. Before measurements, the system was zeroed by adjusting the voltmeter after connecting the reference needle to
the tube normally placed in the mouse nose to complete the
circuit.
Solutions and drugs. The Ringer solutions used to perfuse
the nasal epithelium included HEPES-buffered Ringer with
(in mM) 10 HEPES, pH 7.4, 138 NaCl, 5 KCl, 5 Na2HPO4, 1.8
CaCl2, and 1 MgSO4 and chloride-free HEPES-buffered
Ringer with (in mM) 10 HEPES, pH 7.4, 138 sodium glu-
conate, 5 potassium gluconate, 2.5 Na2HPO4, 3.6 hemicalcium gluconate, and 0.5 MgSO4 where chloride ions were
replaced with gluconate. Drugs added to the Ringer solutions
included amiloride (10⫺4 M; Sigma, St. Louis, MO), forskolin
(10⫺5 M; Calbiochem, La Jolla, CA), glibenclamide (5 ⫻ 10⫺4
M; Calbiochem), 5-nitro-2-(3-phenylpropylamino)benzoic
acid (NPPB; 10⫺4 M; Calbiochem), DIDS (5 ⫻ 10⫺4 M; Calbiochem), diphenylamine-2-carboxylate (DPC; 10⫺3 M; RBI,
Natick, MA), and the Rp diastereomer of adenosine 3⬘,5⬘cyclic monophosphothioate (Rp-cAMPS; 10⫺4 M; BioLog, La
Jolla, CA). Except for forskolin, stock solutions of the drugs
were prepared on the day of the experiment.
Statistics. Data are presented as means ⫾ SE. If an individual mouse was tested multiple times, the data were averaged and presented as a single data point in the group mean.
Statistics were performed using Statview (versions 4.5 and
5.0; SAS Institute, Cary, NC) using either an unpaired Student’s t-test, ANOVA followed by Dunnett’s post hoc test, or
Fisher’s r-to-z covariance analysis as indicated. P ⬍ 0.05 was
considered statistically significant.
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Table 1. Transepithelial nasal potential difference values of wild-type and CF mice
Cftr(⫹/⫹, ⫹/⫺)
Mean PD, mV
SE
n
Cftr(⌬F508/⌬F508)
Baseline
Amiloride
Cl free
Forskolin
Baseline
Amiloride
Cl free
Forskolin
⫺11.1
0.4
19
⫺3.4
0.3
18
⫺15.8
1.0
18
⫺2.0
0.8
18
⫺32.7*
1.2
13
⫺10.6*
0.5
11
1.7*
0.8
11
⫺0.2
0.6
11
Baseline and amiloride values represent the steady state obtained after perfusion with Ringer solution and Ringer solution containing
amiloride, respectively. Cl free and forskolin are the change (⌬) in values calculated as described in MATERIALS AND METHODS after the switch
to chloride-free Ringer solution and the further addition of forskolin, both performed during the continuous presence of amiloride. Cystic
fibrosis (CF) transmembrane conductance regulator (Cftr) (⌬F508/⌬F508) mice and their wild-type littermates [Cftr(⫹/⫹, ⫹/⫺)] tested were
congenic on the C57BL/6J background. n, No. of mice. PD, potential difference. * P ⬍ 0.05, wild type vs. cystic fibrosis by unpaired Student’s
t-test.
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Fig. 1. Transepithelial nasal potential difference traces from a wildtype (A) and a ⌬F508/⌬F508 cystic fibrosis (CF; B) mouse on the
original mixed 129/SvEvs and C57BL/6J genetic background. Time
0, perfusion with Ringer solution and amiloride. The junction potential marks the switch to chloride-free Ringer solution containing
amiloride followed by the switch to chloride-free HEPES-buffered
Ringer (HBR) containing amiloride and forskolin as indicated.
TEPD, transepithelial potential difference. C: recording in which a
bath of HBR was substituted for the mouse. Trace shows that the
junction potential is a consistent function of the circuitry and that
the potential difference is stable after the perfusate was changed to
chloride-free HBR.
With the addition of chloride-free Ringer solution, we
consistently saw a predicted junction potential of approximately ⫺12 mV because of the difference in ion
composition between the chloride-free solution perfusing the nostril and the isotonic solution used in the
reference bridge. In calculating the low chloride response, the initial value used was the peak of the
junction potential. The ⌬TEPD in response to chloridefree solution was also significantly different between
wild-type and CF mice (P ⬍ 0.0001). In wild-type mice,
perfusion of the nasal epithelium with chloride-free
solution caused a hyperpolarization (⌬TEPD ⫽
⫺15.8 ⫾ 1.0 mV). In contrast, the TEPD measured in
CF mice became more depolarized in response to the
chloride-free solution (⌬TEPD ⫽ 1.7 ⫾ 0.8 mV). As
seen in Fig. 1, a similar trend was observed in mixed
C57/129 mice when forskolin was added to the chloride-free Ringer solution to stimulate chloride secretion by increasing the cAMP level. However, the same
phenomenon was not observed with ⌬F508/⌬F508 mice
congenic on the C57BL/6J background and their
non-CF littermates (Table 1). Here the forskolin responses were lower in the wild-type mice, resulting in
no significant (P ⫽ 0.1420) difference between CF and
wild-type mice. The differences in the forskolin response caused by the genetic background are seen
consistently (data not shown) and are being pursued
further.
The nasal TEPD chloride-free and forskolin responses show little correlation. The lack of chloride
transport in CF in response to low chloride challenge or
to stimulation by forskolin suggested that both transport mechanisms may occur through CFTR. It has been
suggested that, in non-CF humans, the hyperpolarization that occurs in response to a chloride gradient is the
result of basal levels of chloride secretion and that the
hyperpolarization with the further addition of isoproterenol is a result of stimulated chloride secretion, both
occurring through CFTR (20). Therefore, it was hypothesized that because of limiting amounts of CFTR
and its regulatory compounds, a larger CFTR basal
chloride response would result in a reduced CFTRstimulated chloride response and vice versa.
We tested this hypothesis in mice by comparing the
changes in PD caused by chloride-free Ringer solution
and those caused by the further addition of forskolin to
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DISTINCT MECHANISMS OF EPITHELIAL CHLORIDE SECRETION
Fig. 2. Comparison of chloride-free and forskolin transepithelial
nasal potential difference values from 6 inbred strains of mice.
Values were calculated after reaching a steady state in the presence
of chloride-free Ringer solution and chloride-free Ringer solution
containing forskolin (n ⫽ 8–10 mice/experiment). Values shown are
mean change (⌬) in TEPD ⫾ SE. Amiloride was present in both
solutions. Mice were exposed to Ringer solution containing amiloride
until a steady state was reached before the switch to chloride-free
Ringer solution.
AJP-Lung Cell Mol Physiol • VOL
Fig. 3. Correlation of chloride-free and forskolin transepithelial nasal potential difference values from the C57BL/6J strain. Values
represent individual mouse ⌬TEPD measurements calculated after
reaching a steady state in the presence of chloride-free Ringer
solution followed by a steady state in chloride-free Ringer solution
containing forskolin (n ⫽ 26 mice). Amiloride was present in both
solutions. Mice were exposed to Ringer solution containing amiloride
until a steady state was reached before the switch to chloride-free
Ringer solution. Solid line represents the mean, and dotted lines
represent the 95% confidence interval for the mean (R2 ⫽ 0.27, P ⫽
0.4297 by Fisher’s r-to-z covariance analysis).
4B), presumed to be mediated by CFTR. DPC reduced
the forskolin response to ⫺1.9 mV compared with the
control (⌬TEPD ⫽ ⫺7.0 mV), whereas DIDS (⌬TEPD ⫽
⫺7.0 mV) had little effect.
Figure 4C summarizes the effects of DIDS, DPC, and
several additional chloride channel inhibitors. Although none of the chloride channel inhibitors eliminate the low chloride response, both glibenclamide and
the combination of DIDS and DPC did significantly
(⌬TEPD: glibenclamide ⫽ ⫺11.0 ⫾ 1.1 mV and DIDS ⫹
DPC ⫽ ⫺10.0 ⫾ 2.2 mV vs. control ⫽ ⫺19.4 ⫾ 0.9 mV;
P ⱕ 0.01) reduce the low chloride response. These same
compounds eliminated the forskolin response (⌬TEPD:
glibenclamide ⫽ 0.2 ⫾ 0.9 mV; P ⱕ 0.01; DIDS ⫹
DPC ⫽ 0.1 ⫾0.8 mV; P ⱕ 0.05 vs. control ⫽ ⫺5.9⫹1.3
mV). Additionally, DPC significantly (⌬TEPD ⫽ ⫺1.4
⫾1.0 mV; P ⱕ 0.05) reduced the forskolin response.
Effect of a protein kinase A inhibitor, Rp-cAMPS, on
the chloride-free and forskolin responses. CFTR is regulated by cAMP-dependent phosphorylation and by
intracellular ATP (1). By inhibiting protein kinase A
(PKA) with Rp-cAMPS, we investigated the effect of
acute PKA inhibition on both the low chloride and
forskolin responses. Figure 5 demonstrates that inhibiting PKA eliminates the forskolin response (⌬TEPD ⫽
⫺6.8 ⫾ 1.6 mV for control and ⫺0.4 ⫾ 1.4 mV for
Rp-cAMPS, P ⫽ 0.015). In contrast, Rp-cAMPS had no
significant effect on the response to low chloride challenge (⌬TEPD ⫽ ⫺18.4 ⫾ 1.4 mV for control and
⫺17.4 ⫾ 1.7 mV for Rp-cAMPS). There was no effect of
Rp-cAMPS on the low chloride response when the
duration of low chloride exposure was extended (data
not shown), suggesting the lack of effect is not an issue
of drug access to the cell.
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stimulate CFTR chloride secretion. We found no correlation for the two responses between several inbred
strains (Fig. 2) or within an individual inbred strain
(Fig. 3). We found that the forskolin response measured in six inbred strains of mice varies little between
the strains. This contrasts with large variations in the
low chloride response measured in the same strains
(Fig. 2). Specifically, the C57BL/6J strain had the most
negative low chloride response (⌬TEPD ⫽ ⫺16.5 ⫾ 0.6
mV), whereas the A/J strain had the least negative low
chloride response (⌬TEPD ⫽ ⫺4.8 ⫾ 0.8 mV) of the
strains tested, and yet their forskolin responses were
nearly identical (⌬TEPD: C57BL/6J, ⫺2.5 ⫾ 1.1 mV
and A/J, ⫺2.6 ⫾ 0.8 mV). Even within a single inbred
strain, in this case C57BL/6J, there was little correlation between low chloride and forskolin responses, as
seen in Fig. 3.
Effect of chloride channel inhibitors on the chloridefree and forskolin responses. The lack of correlation
between the low chloride and forskolin responses
raised the possibility that the low chloride response
may involve channels other than CFTR. To test this
possibility further, we examined the effect of several
chloride channel inhibitors that have been characterized as to their effect on CFTR on the low chloride and
forskolin responses in C57BL/6J mice. Figure 4 shows
the effect of DPC, which is known to inhibit CFTR and
other chloride channels (4, 21, 35, 36, 38), and DIDS,
which is known to inhibit chloride channels other than
CFTR (5, 13, 28, 35). DIDS and DPC have little effect
on the response to a low chloride gradient compared
with that in control mice, as shown in Fig. 4A
(⌬TEPD ⫽ ⫺19.5 mV for control, ⫺17.5 mV for DPC,
and ⫺17.5 mV for DIDS). In contrast, DIDS and DPC
had the expected results on the forskolin response (Fig.
DISTINCT MECHANISMS OF EPITHELIAL CHLORIDE SECRETION
L1177
DISCUSSION
The absence or reduction of functional CFTR is
clearly the fundamental defect in CF. However, it is
not clear how this deficit is involved in the pathophysiology of the disease. Loss of CFTR results in a failure
of the airway epithelium to secrete chloride not only in
response to cAMP but also in response to a chloride
gradient. Concomitantly, there is an increase in sodium absorption across the airway epithelium. The loss
of response to cAMP appears to be the result of the loss
of both CFTR and a channel (ORCC) whose physiological role is unknown, at least in some airway cells. The
ORCC activity appears to be distinct from CFTR in
channel pharmacology as well as in activation and
conductance properties. It is nonetheless CFTR dependent, since CF airway cells heterologously expressing
functional CFTR display ORCC activity in response to
cAMP (27).
Fig. 4. Effect of a panel of chloride channel inhibitors on the chloride-free and forskolin transepithelial nasal potential difference responses of C57BL/6J mice. Mice were assayed for a chloride-free
response (A) and forskolin response (B) in the presence of diphenylamine-2-carboxylate (DPC) or DIDS and in the absence of either
chloride channel blocker. Traces shown are averages ⫾ SE of data
taken at 30-s intervals. ⌬TEPD at the final 30-s time point in the
presence of chloride-free Ringer solution with amiloride was ⫺19.5
mV for control (n ⫽ 17), ⫺17.5 mV for DPC (n ⫽ 13), and ⫺17.5 mV
for DIDS (n ⫽ 11). ⌬TEPD in the presence of chloride-free Ringer
solution with amiloride and forskolin was ⫺7.0 mV for control (n ⫽
17), ⫺1.9 mV for DPC (n ⫽ 12), and ⫺7.0 mV for DIDS (n ⫽ 10). Mice
were pretreated with the appropriate channel blocker in Ringer
solution containing amiloride for at least 2 min before the switch to
chloride-free Ringer solution (time 0, A) and the further switch to
include forskolin (time 0, B). C: summary of changes in the ⌬TEPD
for the chloride-free response (filled bars) and the forskolin response
(open bars) induced by chloride channel blockers. Bars represent the
mean ⌬TEPD, and error bars represent the SE. The number of mice
tested is shown above the bar. One mouse did not survive the
procedure and could not be included in the forskolin, DIDS, and DPC
treatment. NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid.
*P ⬍ 0.05 and **P ⬍ 0.01 vs. control by ANOVA followed by
Dunnett’s post hoc test.
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Fig. 5. Effect of the Rp diastereomer of adenosine 3⬘,5⬘-cyclic monophosphothioate (Rp-cAMPS), an inhibitor of protein kinase A, on the
chloride-free and forskolin transepithelial nasal potential difference
responses. The ⌬TEPD in response to chloride-free Ringer solution
(filled bars) and chloride-free Ringer solution with forskolin (open
bars) was measured in C57BL/6J mice in the presence and absence
of Rp-cAMPS. Bars represent the mean ⌬TEPD, and the error bars
represent the SE. Mice were pretreated (⬃5 min) with Ringer solution, amiloride, and, for the test group, Rp-cAMPS until a steady
state was reached before the chloride-free and forskolin responses
were measured. For those mice receiving Rp-cAMPS, the compound
was contained in the perfusate throughout experiments. The number
of mice tested is shown above the bars. *P ⬍ 0.05 by unpaired t-test.
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AJP-Lung Cell Mol Physiol • VOL
chiometry of cell types is altered in CF and the proportion of cell types responsible for the chloride-free
response are reduced or absent in CF mice. Finally,
CFTR may play a role in regulating signaling in cells of
the epithelium, or its lack may trigger signals that
downregulate the low chloride participants. Previous
studies support this last possibility. We have shown
that nitric oxide synthase-2 levels are decreased in CF
airway epithelia (32). Furthermore, we have demonstrated that decreasing nitric oxide synthase-2 activity
either acutely by pharmacological means or constitutively by knocking out the Nos2 gene reduces the chloride-free response to levels observed in CF mice (8).
The causal link between reduced CFTR and nitric
oxide synthase-2 is not clear but could explain the
CFTR dependence of the low chloride response as one
of the aberrant signaling processes. These and other
possibilities need to be explored further.
The difference in the low chloride response observed
between several inbred mouse strains in this study
permits the genetic dissection of the low chloride response. Studies are thus currently underway to genetically identify the regulatory mechanisms responsible
for the low chloride response. Regardless of the mechanism, if ion transport is contributing to the pathophysiology in CF patients, there is no reason to expect
any one of the processes, lack of cAMP response, lack of
low chloride response, or increased sodium absorption,
to be more important than the other. Consequently, we
need to better understand each of the processes so that
they can be manipulated singularly and jointly to determine their relative roles in the disease process.
This work was supported by National Institutes of Health Specialized Center of Research Grant HL-60293, Core Center Grant
DK-27651, and Training Grant HL-07415.
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The chloride-free response, also absent from CF epithelia, is clearly CFTR dependent at some level. The
mechanism responsible for this basal conductance has
not been identified. A reasonable assumption would be
that it represents basally active CFTR, possibly because of resting levels of cAMP and PKA activities. In
human nasal TEPD studies, it is the combination of the
low chloride and cAMP-stimulated responses that discriminates between CF and non-CF individuals, leading to the postulation that the responses reflect two
activity states of CFTR, a basal state and a stimulated
state (20). If so, one might predict an inverse relationship between the two responses.
The results presented here show that the chloridefree and cAMP responses are distinct. Our murine
studies found a lack of correlation between the chloride-free and cAMP-stimulated responses either within
or between inbred strains of mice. Additionally, we
found that each response had a characteristic pharmacology both to a panel of chloride channel inhibitors
and to a PKA inhibitor that differentiated the two
responses. Although it cannot be excluded that there
are channel activities common to both responses, there
must be activities distinct to each. This suggests that
the response to low chloride challenge may reflect
channels or regulatory processes that are CFTR dependent, analogous to the ORCC properties of single cells,
where CFTR is required for an ORCC-mediated chloride conductance, but neither channel is the sole carrier of the apical epithelial chloride conductance.
In trying to identify the mechanism involved in the
low chloride response, we have attempted to reconcile
with data on known channels. A caveat to that approach is that most channels are characterized at the
channel or cell level to remove the complexities of
intact tissue from the analysis. Tarran et al. (34) have
shown that, even within ciliated cells of mouse nasal
epithelium, there are at least two cell types with regard to unstimulated chloride transport. When one
considers that there are even more epithelial cell types
and that some ion permeation occurs paracellularly,
the intact epithelium is certain to display characteristics not found in cultured or individual primary cells.
So, although the TEPD assay is limited in the detail
with which electrophysiological responses can be described, it does allow for the intact epithelium to be
monitored in vivo.
We propose that the simplest and most consistent
explanation of our data is that the low chloride response is transcellular and involves channels distinct
from CFTR. Furthermore, the same response is virtually absent in CF mice, establishing a requirement of
the low chloride response for CFTR. Although the nature of the requirement for CFTR in the chloride-free
response is unclear, we can propose several modes of
interaction. It is possible that CFTR may dimerize with
other chloride channels such that mutations in CFTR
alter chloride transport through these partner channel(s). Alternatively, the observation that different
subsets of airway epithelial cells express different chloride conductances raises the possibility that the stoi-
DISTINCT MECHANISMS OF EPITHELIAL CHLORIDE SECRETION
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