Departments of 1 Environmental Health Sciences, 3 Physiology and Biophysics, 4 Medicine, 5 Anesthesiology, and 6 Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005; and 2 Department of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies
demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO)
enhances transepithelial Cl secretion by increasing
basolateral K+ conductance (GK)
(Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ.
J Pharmacol Exp Ther 292: 778-787, 2000). Hence
these compounds may be useful to treat cystic fibrosis (CF) airway
disease. The goal of the present study was to determine whether
chlorzoxazone or 1-EBIO altered ion transport across
F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1
monolayers exhibited a basal short-circuit current that was abolished
by apical amiloride (inhibition constant 320 nM) as expected for
Na+ absorption. The addition of chlorzoxazone (400 µM) or
1-EBIO (2 mM) increased the amiloride-sensitive
Isc ~2.5-fold. This overlapping specificity
may preclude use of these compounds as CF therapeutics. Assaying for
changes in the basolateral GK with a
K+ gradient plus the pore-forming antibiotic amphotericin B
revealed that chlorzoxazone or 1-EBIO evoked an ~10-fold increase in
clotrimazole-sensitive GK. In contrast,
chlorzoxazone did not alter epithelial Na+ channel-mediated
currents across basolateral-permeabilized monolayers or in
Xenopus oocytes. These data further suggest that alterations in basolateral GK alone can modulate epithelial
Na+ transport.
epithelial sodium channel; cystic fibrosis transmembrane conductance regulator; clotrimazole; amiloride; 1-ethyl-2-benzimidazolinone
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CYSTIC
FIBROSIS (CF) is a common inherited disease caused by mutations
in the gene coding for a cAMP-regulated epithelial anion channel called
the CF transmembrane conductance regulator (CFTR) (28,
38). CF patients have impaired CFTR-mediated
Cl and HCO
Basolateral K+ conductance (GK) can
play an important role in modulating vectorial Cl
secretion (see Ref. 7 for a review). K+
recycling via basolateral GK is essential to
maintain the mass and charge balance across the
Cl
-secreting epithelial cell. Furthermore, increasing the
GK will hyperpolarize the plasma membrane
potential and thereby increase the driving force for Cl
efflux via CFTR or other apical anion channels. Hence basolateral K+ channels provide a target for therapeutics to overcome
the CF Cl
secretory defect (32). In this
regard, two potential lead compounds have been identified, namely,
1-ethyl-2-benzimidazolinone (1-EBIO) and chlorzoxazone (9, 10,
12, 31). Previous studies (9, 10, 12, 31, 34)
showed that these compounds augment electrogenic Cl
secretion across CFTR-expressing epithelial cells by activating small-
and intermediate-conductance Ca2+-activated K+
channels in the basolateral membranes. Because basolateral
GK is also important in Na+
absorption (7, 35), it is possible that these compounds may increase amiloride-sensitive Na+ absorption. However,
previous reports (6, 8-10, 12, 19, 25, 31, 32) have
focused on the ability of 1-EBIO or chlorzoxazone to enhance
Cl
secretion across cells expressing wild-type CFTR.
The goal of the present study was to quantify the possible effects of
1-EBIO or chlorzoxazone on ion transport across CFT1 airway cells that
are homozygous for the F508 mutation in the CFTR (39).
We grew CFT1 cells as monolayers on filters and mounted them in Ussing
chambers. We observed that the addition of either chlorzoxazone or
1-EBIO evoked an ~2.5- fold increase in amiloride-sensitive Na+ absorption. Next, we investigated the possibility that
the ability of 1-EBIO and chlorzoxazone to increase Na+
absorption was mediated by an increase in basolateral
GK. This was indeed the case. Finally, the
addition of chlorzoxazone to either CFT1 monolayers treated with
basolateral amphotericin B in the presence of an apical-to-basolateral
Na+ gradient or Xenopus oocytes injected with
cRNA for ENaC
-,
-, and
-subunits elicited little enhancement
of the amiloride-sensitive current. Together, these data indicate that
basolateral K+ permeability can play an important role in
modulating active Na+ absorption across airway epithelial
cells. Furthermore, because enhanced Na+ absorption is
thought to contribute to CF airway disease (27), caution
should be observed before administration of these compounds to patients.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
The CFT1 cell line was derived from the trachea of a CF patient and is
homozygous for F508-CFTR (39). The clone 2 CFT1 cells used herein were cultured as previously described (14, 15, 39). Briefly, the cells were maintained in serum-free Ham's
F-12 medium supplemented with seven hormones and growth factors:
insulin (5 µg/ml), endothelial cell growth supplement (3.7 µg/ml),
transferrin (5 µg/ml), epidermal growth factor (25 ng/ml),
triiodothyronine (3 × 10
8 M), hydrocortisone
(1 × 10
6 M), and cholera toxin (10 ng/ml)
(15). Before being seeded on filters, CFT1 cells were
passaged into serum-free F-12 medium formulated without cholera toxin.
Confluent monolayers for Ussing chamber experiments were obtained by
plating the cells on collagen-coated filter inserts (Falcon, Becton
Dickinson, Franklin Lakes, NJ) at a density of 1.2 × 105 cells/cm2. On the day after seeding, the
culture medium was replaced with the Ham's F-12 medium without cholera
toxin diluted 1:1 with NIH/3T3 cell-conditioned DMEM containing 2%
fetal bovine serum. Ussing chamber experiments were performed 7-10
days after seeding because a previous study (15) indicated
that the transepithelial resistance was maximal during this period
(15).
Epithelial voltage clamp. Filter inserts containing confluent monolayers were mounted in modified Ussing chambers (Jim's Instrument, Iowa City, IA). Unless otherwise specified, the cells were bathed on both sides with identical HEPES-buffered saline solutions containing (in mM) 128 NaCl, 5 sodium pyruvate, 4 KCl, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 5 HEPES-NaOH (pH 7.4). The bath temperature was maintained at 37°C, and both solutions were stirred and gassed with room air. Short-circuit current (Isc) measurements were obtained with an epithelial voltage clamp (Physiologic Instruments VCC600, San Diego, CA) as previously described (1, 35, 36). A voltage pulse was imposed every 100 s to monitor the transepithelial resistance, which was calculated with Ohm's law. Amiloride (100 µM) was added to the apical solution to abolish the Isc component due to Na+ absorption (3). In one series of experiments, amiloride was added apically in increasing concentrations from 1 nM to 30 µM to determine the affinity of amiloride for the underlying Na+ channel.
In experiments designed to assay for changes in basolateral GK, transepithelial ion gradients were created by replacing the apical bath with a high-K+ solution that contained (in mM) 132 potassium gluconate, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 5 HEPES-KOH (pH 7.4). Most of the apical bath ClRNA synthesis.
The pSPORT plasmid (GIBCO BRL, Life Technologies, Gaithersburg, MD)
containing either rat -,
-, or
-ENaC (a generous gift from Dr.
B. Rossier, University of Lausanne, Lausanne, Switzerland) was
linearized by overnight incubation with NotI (Promega,
Madison, WI), digested with 1% SDS and 200 µg/ml of proteinase K for
60 min at 50°C, extracted with phenol-chloroform, and precipitated. Full-length wild-type CFTR contained in pBluescript was treated similarly, with the exception that the plasmid was linearized with
EcoRV. Sense RNA from each transcript was in vitro
transcribed from precipitated plasmid DNA with a T7 mMessage Machine
kit according to the manufacturer's instructions (Ambion, Austin, TX).
The integrity and size of the resulting cRNAs were verified by
electrophoresis through denaturing 1% agarose-formaldehyde gels.
Expression of ENaC or CFTR in oocytes and two-electrode voltage clamp. These methods have been previously described in detail (13). Briefly, partial ovariectomies were performed on anesthetized female Xenopus laevis toads (Xenopus Express, Beverly Hills, FL). Oocytes were manually defolliculated and incubated overnight in L15 medium diluted 1:1 with deionized water supplemented with penicillin (200 U/ml) and streptomycin (200 µg/ml). On the following day, the oocytes were injected with 8.3 ng of each rat ENaC subunit cRNA (25 ng of total cRNA) or 25 ng of CFTR cRNA in a 50-nl volume with a Nanoject microinjector (Drummond, Broomall, PA). Currents were evaluated 36-48 h after injection with a two-electrode voltage clamp (Geneclamp 500, Axon Instruments, Foster City, CA). The oocytes were bathed in a solution containing (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 0.2 CaCl2, and 15 HEPES-NaOH (pH 7.6). Voltage was clamped to 0 mV, and inward and outward currents were recorded at 20-s intervals at holding potentials of ±100 mV. The amiloride-sensitive current was calculated by subtracting the inward current recorded after the addition of 10 µM amiloride from that before the addition.
Chemicals. Amphotericin B and 1-EBIO were the generous gifts from Drs. S. Lucania (Bristol-Myers Squibb, Princeton, NJ) and J. Tomich (Kansas State University, Manhattan, KS), respectively. Amiloride and clotrimazole were purchased from Sigma. Chlorzoxazone was obtained from Aldrich. All drugs were added to the Ussing chamber solutions in a small volume of concentrated stock. Stock solutions of 1-EBIO (0.1 M), chlorzoxazone (0.1 M), and amphotericin B (3 mM) were prepared in DMSO. Clotrimazole and amiloride stocks were made in ethanol (10 mM) and water (10 µM to 10 mM), respectively. Because of their lipophilic nature, 1-EBIO, chlorzoxazone, and clotrimazole were added to both sides of the monolayers (10, 11, 31).
Data analysis.
The data are means ± SD; n is the number of different
experiments. Curve fits were performed with the least squares routine in SigmaPlot (version 5.0 for Windows, SPSS Science). All dose-response relationships were initially fit to single-site Michaelis-Menten functions for activation {Isc = (Isc)max · [D]/([D] + Ks)} or inhibition
{Isc = (Isc)max · Ki/([D] + Ki)}, where
(Isc)max is the maximal
Isc and the equilibrium constants
Ks and Ki represent the
concentration of drug ([D]) causing 50% stimulation or 50% inhibition, respectively. The Hill equation
Isc = (Isc)max · [
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chlorzoxazone or 1-EBIO increases amiloride-sensitive
Isc across CFT1 monolayers.
Filters containing CFT1 cells were mounted in Ussing chambers and
bathed on both sides with standard HEPES-buffered saline solutions.
CFT1 monolayers exhibited a basal or resting Isc
of 4.0 ± 1.5 µA/cm2 and transepithelial resistance
of 119 ± 22 · cm2 (n = 13). Figure 1 compares a representative
current record after chlorzoxazone addition to a paired control. The
addition of 600 µM chlorzoxazone caused the
Isc across intact cells to increase ~2.5-fold
(from 3.6 ± 1.5 to 9.4 ± 1.0 µA/cm2;
n = 7). Subsequent addition of the Na+
channel blocker amiloride (100 µM) to both apical bath solutions abolished the basal and chlorzoxazone-stimulated currents. Active Na+ absorption mediated the Isc
response to chlorzoxazone because it was eliminated by amiloride
pretreatment (data not shown; n = 4; see Ref.
14). 1-EBIO was less potent than chlorzoxazone, requiring
2 mM to evoke a similar enhancement in amiloride-sensitive Isc (Fig.
2A). On average, the
addition of 2 mM 1-EBIO caused Isc to
increase from 4.4 ± 1.4 to 10.5 ± 2.7 µA/cm2
(n = 6). To quantify the dose-response relationships,
we added 1-EBIO or chlorzoxazone in increasing concentrations as
illustrated by the representative tracing shown in Fig. 2A.
These data are summarized in Fig. 2B. Both data sets were
poorly described by simple Michaelis-Menten activation functions, and
the curves in Fig. 2B depict the least squares fits to the
Hill equation (30). These data show that chlorzoxazone
(Ks = 330 ± 21 µM) was a slightly more potent activator of Na+ absorption than 1-EBIO
(Ks = 444 ± 48 µM). This
Ks for 1-EBIO is similar to the value (490 µM)
previously reported for the enhancement of Cl
secretion
(10). The plots also show a twofold difference in the Hill
coefficients for chlorzoxazone (nH = 5) vs.
1-EBIO (nH = 2.6). This analysis yields
some mechanistic insights that are considered in
DISCUSSION.
|
|
|
Chlorzoxazone or 1-EBIO augments the basolateral GK in
CFT1 cells.
Previous studies indicated that 1-EBIO and chlorzoxazone enhance
transepithelial Cl secretion by increasing the
basolateral GK. Because active Na+
absorption also depends on the GK of the
basolateral membranes (7, 35), it is likely that these
compounds alter Na+ absorption by a similar mechanism. We
tested this hypothesis using the technique described by Germann and
colleagues (17, 18), Kirk and Dawson (21),
and Venglarik and Dawson (35) where a
K+ gradient is imposed across the monolayer and 10 µM
amphotericin B is added to eliminate the apical membrane a barrier to
the flow of monovalent cations. Figure 4
shows representative current records. An apical-to-basolateral
K+ gradient was imposed across both monolayers before the
start of the recordings as described in MATERIALS AND
METHODS. Amphotericin B was then added to the apical bathing
solution, which revealed a modest basal current. The subsequent
addition of chlorzoxazone (500 µM) to both sides of one monolayer
caused the Isc to increase approximately
sevenfold. Addition of the K+ channel blocker clotrimazole
(100 µM) to both bathing solutions eliminated most of the basal and
chlorzoxazone-stimulated currents as illustrated by the representative
traces in Fig. 4. 1-EBIO evoked similar increases in
Isc as chlorzoxazone, although higher concentrations were required (Fig. 5).
The concentration-response relationships for
Isc activation were determined by exposing
monolayers to increasing concentrations of 1-EBIO or chlorzoxazone as
illustrated by the representative current trace shown in Fig.
5A. These data are summarized in Fig. 5B.
Clotrimazole (100 µM) was used to block and thereby identify the
channel-mediated K+ current (IK).
The curves show the best fits to the Hill equation by nonlinear
regression. Chlorzoxazone was a slightly more potent stimulator
(Ks = 273 ± 5 µM) and had a
twofold larger Hill coefficient (nH = 3.5)
than 1-EBIO (Ks = 1,060 ± 282 µM;
nH = 1.7). These results generally agree
with the dose dependence of Isc activation in
intact monolayers shown in Fig. 2. Oddly, both chlorzoxazone and 1-EBIO
exerted a biphasic effect on IK, increasing it
at low concentrations while inhibiting it at higher concentrations.
This complicated dose dependency shows that it is important to compare the efficacy between K+ channel activators over a range of
concentrations. Previously, 1-EBIO was reported to be more potent than
chlorzoxazone based on the response to 1 mM chlorzoxazone
(31). Although this conclusion is consistent with the
results obtained at 1 mM chlorzoxazone as shown in Fig. 5B,
our data show that chlorzoxazone was more potent than 1-EBIO throughout
the lower concentration range.
|
|
|
Effect of chlorzoxazone on ENaC- or CFTR-mediated currents in
oocytes.
Previous studies (9, 31) showed that 1-EBIO and
chlorzoxazone enhance transepithelial Cl secretion by
increasing both apical CFTR-mediated Cl
conductance and
basolateral GK. Thus we considered the
possibility that these compounds may also increase the apical ENaC
channel activity in CFT1 cells. Initially, we tested for possible
effects of chlorzoxazone on Isc across CFT1
monolayers exposed to an apical-to-basolateral Na+ gradient
and treated with basolateral amphotericin B (200 µM). In these
experiments, we observed a small Isc (3.1 µA/cm2) that was amiloride sensitive, and chlorzoxazone
addition (400 µM) had no effect (n = 3). However, as
a further test for possible activation of ENaC by chlorzoxazone, we
expressed rat ENaC
-,
-, and
-subunits in Xenopus
oocytes and measured the amiloride-sensitive currents before and after
chlorzoxazone. Oocytes expressing human wild-type CFTR were used as a
control. The results are summarized in Fig.
7. Figure 7A compares
amiloride-sensitive inward current (Iin)
in ENaC-injected oocytes (n = 5) with the inward
current from cAMP-stimulated CFTR-injected oocytes (n = 3) before and after the addition of chlorzoxazone (500 µM).
Chlorzoxazone had only a small effect on the amiloride-sensitive
currents while significantly increasing the current in CFTR-injected
oocytes. The ability of chlorzoxazone to increase CFTR-mediated current is consistent with previous reports (9, 31). Figure
7B shows the dose response for CFTR activation as measured
by the change in (
) Iin. The solid
line in Fig. 7B shows the best fit of the data to a Hill plot, which yielded a Ks of
377 ± 19 µM and a nH of 2.76 ± 0.4.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The data presented here demonstrate that the addition of
chlorzoxazone or 1-EBIO augments Na+ absorption across
immortalized CF airway cells and that this response is mediated by an
~10-fold increase in basolateral GK. Although
it is possible that chlorzoxazone or 1-EBIO may also increase apical
Na+ channel activity, we consider it unlikely for two
reasons. First, chlorzoxazone (500 µM) did not alter the
amiloride-sensitive currents across CFT1 monolayers, with an
apical-to-basolateral Na+ gradient in the presence of
basolateral amphotericin B. Second, chlorzoxazone (500 µM) did not
affect the amiloride-sensitive currents in Xenopus oocytes
expressing -,
-, and
-subunits of the ENaC. In contrast,
chlorzoxazone increased Cl
current across oocytes
expressing CFTR. This enhancement of CFTR-mediated current is expected
based on the known effects of these drugs on CFTR in permeabilized
monolayers (9, 31) and also on the ability of these drugs
to activate the K+ channels expressed in Xenopus
oocytes (34). Thus we consider it likely that the ability
of chlorzoxazone or 1-EBIO to augment Na+ absorption is
mediated entirely by the increase in basolateral GK. This hypothesis is consistent with the
reported effects of chlorzoxazone and 1-EBIO on basolateral
K+ channels in Cl
-secretory epithelial cells.
It is also consistent with the obligatory coupling of apical
Na+ and basolateral K+ currents imposed by the
Koefoed-Johnson Ussing model for Na+ absorption (22; for a
review, see Ref. 7).
Because the apical membrane resistance is 4- to 10-fold higher than that of the basolateral membrane, it is often assumed that changes in amiloride-sensitive Na+ channel activity alone account for agonist-induced alterations in epithelial Na+ absorption (2, 4, 33). This notion would be true if resistance were the only element determining the rate of ionic flow across the two plasma membranes in series. However, driving forces also play a crucial role. To maintain steady-state conditions, the higher resistance of the apical membrane must be precisely balanced by an increased electrochemical gradient for Na+ entry. This difference in driving force arises in part from the cell membrane potential, which favors Na+ entry and opposes K+ exit. Hence both membranes play an important role in determining the rate of transepithelial Na+ absorption (for a review, see Ref. 7). For example, Na+ absorption can be inhibited by K+ channel blockers such as Ba2+ (35) and clotrimazole (Venglarik, unpublished observations). Our data provide additional support for this notion by showing that known K+ channel activators promote transepithelial Na+ absorption across CFT1 airway cells. Chlorzoxazone and 1-EBIO should provide further insights regarding the role of basolateral GK in regulating Na+ absorption in future studies.
Furthermore, the observation that chlorzoxazone and 1-EBIO can increase
Na+ absorption in addition to activating Cl
secretion also agrees with earlier studies (23, 24)
showing that ENaC- and CFTR-mediated currents are present
simultaneously in whole cell recordings of airway epithelial cells. If
ENaC and CFTR are expressed in the same cell or in electrically coupled cells, then K+ channel openers such as chlorzoxazone or
1-EBIO are expected to increase both Na+ absorption and
Cl
secretion (7). Although there is some
evidence that 1-EBIO increases Na+ absorption across
primary cultures of human bronchial epithelial cells (8),
most previous reports (6, 8-10, 12, 19, 25, 31, 32)
have focused on the ability of these compounds to enhance
Cl
secretion via wild-type CFTR. In this regard, the CFT1
cell line (39) provides a unique model to study airway
epithelial Na+ absorption without the confounding effects
of CFTR-mediated anion secretion. Furthermore, data obtained with CFT1
cells are expected to be more relevant to CF patients. Because
Na+ hyperabsorption may contribute to CF airway disease
(27), our data suggest that caution should be observed
before administering chlorzoxazone, 1-EBIO, or related compounds
(32) to CF patients.
Finally, the mechanisms mediating K+ channel activation by benzimidazolinones or benzoxazolones remain unknown. However, our analysis of the dose-response relationships provides some insight. Specifically, all the activation data were described by Hill plots with coefficients > 1. This implies some form of signal amplification. In the case of hemoglobin, there are multiple binding sites that cooperatively increase affinity on increased occupancy (20). However, the Hill model per se cannot account for the approximately twofold difference in coefficients between 1-EBIO and chlorzoxazone. Together, our data indicate that K+ channel activation is mediated by a mechanism involving variable cooperativity or signal amplification that depends on the ligand. A recent patch-clamp study (34) further suggests that this activation mechanism depends on but is not mediated by Ca2+. Finally, the similarity in dose dependence for chlorzoxazone activation of Na+-absorptive current across CFT1 cells (Fig. 2B) and CFTR-mediated current in Xenopus oocytes (Fig. 7B) is suggestive of a common binding site and mechanism. This hypothesis needs to be tested in future studies.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Lan Chen for assistance with oocyte injection and recording. We thank Drs. S. Lucania (Bristol-Myers Squibb), J. Tomich (Kansas State University), and B. Rossier (University of Lausanne, Lausanne, Switzerland) for their gifts of amphotericin B, 1-ethyl-2-benzimidazolinone, and epithelial sodium channel cDNAs, respectively. Finally, we thank Dr. Kevin Kirk for critiquing an earlier version of this article.
![]() |
FOOTNOTES |
---|
This work was supported by the Cystic Fibrosis Foundation (RDP464), the Webb Foundation, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54781, and National Heart, Lung, and Blood Institute Grants HL-46943, HL-37556, HL-31197, and HL51173.
Address for reprint requests and other correspondence: C. J. Venglarik, Dept. of Environmental Health Sciences, Univ. of Alabama at Birmingham, 760 McCallum, 1918 University Blvd., Birmingham, AL 35294 (E-mail: cjv{at}uab.edu).
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 13 February 2001; accepted in final form 21 June 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bebok, Z,
Tousson A,
Schwiebert LM,
and
Venglarik CJ.
Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers.
Am J Physiol Cell Physiol
280:
C135-C145,
2001
2.
Benos, DJ,
Cunningham S,
Baker RR,
Beason KB,
Oh Y,
and
Smith PR.
Molecular characteristics of amiloride-sensitive sodium channels.
Rev Physiol Biochem Pharmacol
120:
31-113,
1992[ISI][Medline].
3.
Benos, DJ,
Simon SA,
Mandel LJ,
and
Cala PM.
Effect of amiloride and some of its analogues of cation transport in isolated frog skin and thin lipid membranes.
J Gen Physiol
68:
43-63,
1976[Abstract].
4.
Blazer-Yost, BL,
Record RD,
and
Oberleithner H.
Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line.
Pflügers Arch
432:
685-691,
1996[ISI][Medline].
5.
Boat, TF,
and
Cheng PW.
Epithelial cell dysfunction in cystic fibrosis: implications for airways disease.
Acta Paediatr Scand Suppl
363:
25-29,
1989[Medline].
6.
Cuthbert, AW,
Hickman ME,
Thorn P,
and
MacVinish LJ.
Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone.
Am J Physiol Cell Physiol
277:
C111-C220,
1999
7.
Dawson, DC,
and
Richards NW.
Basolateral K conductance: role in regulation of NaCl absorption and secretion.
Am J Physiol Cell Physiol
259:
C181-C195,
1990
8.
Devor, DC,
Bridges RJ,
and
Pilewski JM.
Pharmacological modulation of ion transport across wild-type and F508 CFTR-expressing human bronchial epithelia.
Am J Physiol Cell Physiol
279:
C461-C479,
2000
9.
Devor, DC,
Singh AK,
Bridges RJ,
and
Frizzell RA.
Modulation of Cl secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK.
Am J Physiol Lung Cell Mol Physiol
271:
L785-L795,
1996
10.
Devor, DC,
Singh AK,
Frizzell RA,
and
Bridges RJ.
Modulation of Cl secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel.
Am J Physiol Lung Cell Mol Physiol
271:
L775-L784,
1996
11.
Devor, DC,
Singh AK,
Gerlach AC,
Frizzell RA,
and
Bridges RJ.
Inhibition of intestinal Cl secretion by clotrimazole: direct effect on basolateral membrane K+ channels.
Am J Physiol Cell Physiol
273:
C531-C540,
1997
12.
Devor, DC,
Singh AK,
Lambert LC,
DeLuca A,
Frizzell RA,
and
Bridges RJ.
Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.
J Gen Physiol
113:
743-760,
1999
13.
DuVall, MD,
Zhu S,
Fuller CM,
and
Matalon S.
Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing -rENaC.
Am J Physiol Cell Physiol
274:
C1417-C1423,
1998
14.
Gao, L,
Broughman JR,
Iwamoto T,
Tomich JM,
Venglarik CJ,
and
Forman HJ.
Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.
Am J Physiol Lung Cell Mol Physiol
281:
L24-L30,
2001
15.
Gao, L,
Kim KJ,
Yankaskas JR,
and
Forman HJ.
Abnormal glutathione transport in cystic fibrosis airway epithelia.
Am J Physiol Lung Cell Mol Physiol
277:
L113-L118,
1999
16.
Garcia, ML,
Hanner M,
Knaus HG,
Koch R,
Schmalhofer W,
Slaughter RS,
and
Kaczorowski GJ.
Pharmacology of potassium channels.
Adv Pharmacol
39:
425-471,
1997[Medline].
17.
Germann, WJ,
Ernst SA,
and
Dawson DC.
Resting and osmotically induced basolateral K conductances in turtle colon.
J Gen Physiol
88:
253-274,
1986[Abstract].
18.
Germann, WJ,
Lowy ME,
Ernst SA,
and
Dawson DC.
Differentiation of two distinct K conductances in the basolateral membrane of turtle colon.
J Gen Physiol
88:
237-251,
1986[Abstract].
19.
Hamilton, KL,
Meads L,
and
Butt AG.
1-EBIO stimulates Cl secretion by activating a basolateral K+ channel in the mouse jejunum.
Pflügers Arch
439:
158-166,
1999[ISI][Medline].
20.
Hill, AV.
The combinations of haemoglobin with oxygen and carbon monoxide.
Biochem J
7:
471-480,
1913.
21.
Kirk, KL,
and
Dawson DC.
Basolateral potassium channel in turtle colon. Evidence for single-file ion flow.
J Gen Physiol
82:
297-329,
1983[Abstract].
22.
Koefoed-Johnsen, V,
and
Ussing HH.
The nature of the frog skin potential.
Acta Physiol Scand
42:
298-308,
1958[ISI].
23.
Kunzelmann, K,
Kathofer S,
and
Greger R.
Na+ and Cl conductances in airway epithelial cells: increased response to amiloride and Na+ replacement in cystic fibrosis.
Pflügers Arch
431:
1-9,
1995[ISI][Medline].
24.
Kunzelmann, K,
Kathofer S,
Hipper A,
Gruenert DC,
and
Gregner R.
Culture-dependent expression of Na+ conductances in airway epithelial cells.
Pflügers Arch
431:
578-586,
1996[ISI][Medline].
25.
MacVinish, LJ,
Hickman ME,
Mufti DA,
Durrington HJ,
and
Cuthbert AW.
Importance of basolateral K+ conductance in maintaining Cl secretion in murine nasal and colonic epithelia.
J Physiol (Lond)
510:
237-247,
1998
26.
Pedersen, KA,
Schroder RL,
Skaaning-Jensen B,
Strobaek D,
and
Olesen SP.
Activation of the human intermediate-conductance Ca2+-activated K+ channel by 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent.
Biochim Biophys Acta
1420:
231-240,
1999[ISI][Medline].
27.
Pilewski, JM,
and
Frizzell RA.
Role of CFTR in airway disease.
Physiol Rev
79:
S215-S255,
1999[Medline].
28.
Riordan, JR,
Rommens JM,
Kerem B,
Alon N,
Rozmahel R,
Grzelczak Z,
Zielenski J,
Lok S,
Plavsic N,
Chou JL,
Drumm MJ,
Iannuzzi MC,
Collins FS,
and
Tsui L.
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.
Science
245:
1066-1073,
1989[ISI][Medline].
29.
Rufo, PA,
Jiang L,
Moe SJ,
Brugnara C,
Alper SL,
and
Lencer WI.
The antifungal antibiotic, clotrimazole, inhibits Cl secretion by polarized monolayers of human colonic epithelial cells.
J Clin Invest
98:
2066-2075,
1996
30.
Segel, IH.
Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry (2nd ed.). New York: Wiley, 1976.
31.
Singh, AK,
Devor DC,
Gerlach AC,
Gondor M,
Pilewski JM,
and
Bridges RJ.
Stimulation of Cl secretion by chlorzoxazone.
J Pharmacol Exp Ther
292:
778-787,
2000
32.
Singh, S,
Syme CA,
Singh AK,
Devor DC,
and
Bridges RJ.
Benzimidazolone activators of chloride secretion: potential therapeutics for cystic fibrosis and chronic obstructive pulmonary disease.
J Pharmacol Exp Ther
296:
600-611,
2001
33.
Stutts, MJ,
Canessa CM,
Olsen JC,
Hamrick M,
Cohn JA,
Rossier BC,
and
Boucher RC.
CFTR as a cAMP-dependent regulator of sodium channels.
Science
269:
847-850,
1995[ISI][Medline].
34.
Syme, CA,
Gerlach AC,
Singh AK,
and
Devor DC.
Pharmacological activation of cloned intermediate- and small-conductance Ca2+-activated K+ channels.
Am J Physiol Cell Physiol
278:
C570-C581,
2000
35.
Venglarik, CJ,
and
Dawson DC.
Cholinergic regulation of Na absorption by turtle colon: role of basolateral K conductance.
Am J Physiol Cell Physiol
251:
C563-C570,
1986
36.
Venglarik, CJ,
Schultz BD,
DeRoos AD,
Singh AK,
and
Bridges RJ.
Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl channels.
Biophys J
70:
2696-2703,
1996[Abstract].
37.
Warncke, J,
and
Lindemann B.
Voltage dependence of the blocking rate constants of amiloride at apical Na channels.
Pflügers Arch
405:
S89-S94,
1985[ISI][Medline].
38.
Welsh, MJ,
and
Smith AE.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[ISI][Medline].
39.
Yankaskas, JR,
Haizlip JE,
Conrad M,
Koval D,
Lazarowski E,
Paradiso AM,
Rinehart CA, Jr,
Sarkadi B,
Schlegel R,
and
Boucher RC.
Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.
Am J Physiol Cell Physiol
264:
C1219-C1230,
1993