Departments of 1 Cell Biology and Physiology and 2 Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Forskolin,
UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen
(Methoxsalen; 8-MOP), and genistein were evaluated for their
effects on ion transport across primary cultures of human bronchial
epithelium (HBE) expressing wild-type (wt HBE) and F508
(
F-HBE) cystic fibrosis transmembrane conductance regulator. In wt
HBE, the baseline short-circuit current (Isc)
averaged 27.0 ± 0.6 µA/cm2 (n = 350). Amiloride reduced this Isc by 13.5 ± 0.5 µA/cm2 (n = 317). In
F-HBE,
baseline Isc was 33.8 ± 1.2 µA/cm2 (n = 200), and amiloride reduced
this by 29.6 ± 1.5 µA/cm2 (n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE,
subsequent to amiloride, forskolin induced a sustained,
bumetanide-sensitive Isc
(
Isc = 8.4 ± 0.8 µA/cm2; n = 119). Addition of
acetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid further reduced Isc, suggesting forskolin also stimulates
HCO3
secretion. This was confirmed by ion
substitution studies. The forskolin-induced Isc
was inhibited by 293B, Ba2+, clofilium, and quinine,
whereas charybdotoxin was without effect. In
F-HBE the forskolin
Isc response was reduced to 1.2 ± 0.3 µA/cm2 (n = 30). In wt HBE, mucosal UTP
induced a transient increase in Isc (
Isc = 15.5 ± 1.1 µA/cm2;
n = 44) followed by a sustained plateau, whereas in
F-HBE the increase in Isc was reduced to
5.8 ± 0.7 µA/cm2 (n = 13). In wt
HBE, 1-EBIO, NS004, 8-MOP, and genistein increased Isc by 11.6 ± 0.9 (n = 20), 10.8 ± 1.7 (n = 18), 10.0 ± 1.6 (n = 5), and 7.9 ± 0.8 µA/cm2
(n = 17), respectively. In
F-HBE, 1-EBIO, NS004, and
8-MOP failed to stimulate Cl
secretion. However, addition
of NS004 subsequent to forskolin induced a sustained Cl
secretory response (2.1 ± 0.3 µA/cm2,
n = 21). In
F-HBE, genistein alone stimulated
Cl
secretion (2.5 ± 0.5 µA/cm2,
n = 11). After incubation of
F-HBE at 26°C for
24 h, the responses to 1-EBIO, NS004, and genistein were all
potentiated. 1-EBIO and genistein increased Na+ absorption
across
F-HBE, whereas NS004 and 8-MOP had no effect. Finally,
Ca2+-, but not cAMP-mediated agonists, stimulated
K+ secretion across both wt HBE and
F-HBE in a
glibenclamide-dependent fashion. Our results demonstrate that
pharmacological agents directed at both basolateral K+ and
apical Cl
conductances directly modulate Cl
secretion across HBE, indicating they may be useful in ameliorating the
ion transport defect associated with CF.
cystic fibrosis; 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one; genistein; 1-ethyl-2-benzimidazolinone; 8-methoxypsoralen; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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THE HALLMARK ION TRANSPORT DEFECTS
in cystic fibrosis (CF) are a diminished or absent Cl
secretory response to cAMP-mediated agonists and Na+
hyperabsorption. The most common mutation in the CF transmembrane conductance regulator (CFTR) gene product, a deletion of phenylalanine at amino acid 508 (
F508), is particularly insidious, in that it
leads to both a defect in the trafficking of the mutant protein to the
apical membrane (55) as well as a channel that exhibits defective gating (1, 44). Pharmacologically,
there are theoretically several means to ameliorate this primary ion
transport defect. First, one can correct the trafficking defect
associated with
F508 CFTR (5). This approach was
highlighted by Denning et al. (9) who demonstrated
that decreasing the temperature at which
F508 CFTR-expressing cells
are grown increases the trafficking of the mutant protein to the plasma membrane.
A second strategy is to develop agents capable of directly interacting
with any F508 CFTR expressed in the apical membrane. Highlighting
this possibility, Kalin et al. (31) recently
demonstrated that
F508 CFTR can be expressed at the apical
membrane of CF airway. The benzimidazolone,
5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one (NS004) was the first compound shown to activate both wild-type and
F508 CFTR in excised patch-clamp recordings (23).
However, we demonstrated that neither NS004 nor its structurally
related analog, NS1619, stimulated Cl
secretion in either
the T84 cell line or in primary cultures of murine tracheal epithelium
(MTE), despite the fact that these compounds increased apical membrane
Cl
conductance (13). Nguyen et al.
(41) first demonstrated that the flavones quercetin and
kaempferol stimulated Cl
secretion across the T84 model
secretory epithelium in a cAMP-independent manner. Subsequently, it has
been shown that the related compound, genistein, also stimulates
Cl
secretion (28). More recent evidence
indicates that genistein directly interacts with CFTR to increase the
open probability of the channel (25, 54).
Finally, one can bypass the CFTR defect altogether by modulating the
activity of alternative ion conductive pathways. For example,
increasing intracellular Ca2+ has been shown to stimulate
Cl secretion across CF airway (57). Indeed,
Mason et al. (37) demonstrated that the
Ca2+-dependent agonist, UTP, acting at P2y2
receptors, stimulates Cl
secretion across CF tracheal
epithelium. We previously characterized the basolateral membrane
K+ channel activated by Ca2+-mediated agonists
(KCa) in colonic and airway epithelia (10, 16). We demonstrated that direct pharmacological
activation of KCa by the benzimidazolone,
1-ethyl-2-benzimidazolinone (1-EBIO), resulted in the stimulation of
Cl
secretion across T84 and Calu-3 cells as well as
primary cultures of MTE (13, 15). These
results suggest that basolateral K+ channels may represent
unique pharmacological targets for CF therapy (13,
15, 16).
Although 1-EBIO, genistein, 8-methoxypsoralen (Methoxsalen; 8-MOP), and
NS004 have been shown to stimulate Cl secretion across
T84 cells and MTE, the effects of these compounds on Cl
secretion across human airway have not been evaluated. Therefore, we
determined the effects of these agonists on primary cultures of human
bronchial epithelia (HBE) expressing wild-type (wt HBE) or
F508
(
F-HBE) CFTR. We demonstrate that 1-EBIO, NS004, 8-MOP, and
genistein stimulate a sustained Cl
secretory response in
wt HBE. In
F-HBE both NS004 and genistein stimulate a small
Cl
secretory response, whereas 1-EBIO and 8-MOP do not.
Additionally, following incubation of the cells at 26°C, the
responses to 1-EBIO, NS004, and genistein are all potentiated. These
results indicate that an apical membrane Cl
conductance,
perhaps
F508 CFTR, is expressed and can be pharmacologically modulated in
F-HBE. Further, these results demonstrate that, despite
the low levels of expression of CFTR in native tissue, pharmacological
agents directed at either apical Cl
or basolateral
K+ channels are capable of modulating Cl
secretion, supporting the notion that they may be therapeutically useful for CF.
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METHODS |
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Primary Cultures of HBE
HBE was obtained from excess pathological tissue remaining after lung transplantation under a protocol approved by the University of Pittsburgh Investigational Review Board. Tissue expressing wt CFTR was obtained following lung transplantation for a variety of pathological conditions including bronchiectasis, emphysema, primary pulmonary hypertension, pulmonary fibrosis, andIsc Measurements
Costar Transwell cell culture inserts were mounted in an Ussing chamber (Jim's Instruments, Iowa City, IA), and the monolayers were continuously short circuited (University of Iowa, Department of Bioengineering). Transepithelial resistance was measured by periodically applying a 5-mV pulse and the resistance calculated using Ohm's law. The bath solution contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of this solution was 7.4 when gassed with a mixture of 95% O2-5% CO2 at 37°C. In zero ClChemicals
NS004 was a generous gift from Dr. Soren Peter-Olesen (Neurosearch, Glostrup, Denmark), 293B was a generous gift from Dr. Rainer Greger (Albert-Ludwigs-Universtat, Freiberg, Germany), and nystatin was a generous gift from Dr. S. Lucania (Bristol Meyers-Squibb). 1-EBIO was obtained from Aldrich Chemical; genistein was obtained from Indofine Chemical (Somerville, NJ); UTP was obtained from Calbiochem (La Jolla, CA); bumetanide, quinine, 8-MOP, and forskolin were obtained from Sigma Chemical (St. Louis, MO). 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and clofilium were obtained from RBI (Natick, MA). DNDS was obtained from Pfaltz and Bauer (Westbury, CT). Charybdotoxin (CTX) was obtained from Accurate Chemical and Scientific (Westbury, NY). All compounds, prepared in either DMSO or ethanol, were made asData Analysis
All data are presented as means ± SE, where n indicates the number of experiments. Apparent inhibitory (Ki) and stimulatory (Ks) constants were obtained using nonlinear curve-fitting routines in SigmaPlot (Jandel Scientific, San Rafael, CA). Statistical analysis was performed using Student's t-test. A value of P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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In total, we evaluated 350 wt CFTR-expressing HBE monolayers from
40 patients under short-circuit conditions in symmetric standard bath
solution. The baseline Isc averaged 27.0 ± 0.6 µA/cm2 with a transepithelial potential difference
(PDte) and resistance (Rte) of
-15.6 ± 0.5 mV and 646 ± 18 · cm2,
respectively. Addition of amiloride (10 µM) to the mucosal chamber reduced Isc an average of 13.5 ± 0.5 µA/cm2 to a new plateau value of 13.5 ± 0.4 µA/cm2 (n = 317). In contrast, in 200 monolayers, from 16 patients homozygous for the
F508 CFTR
mutation (
F-HBE), the baseline Isc
averaged 33.8 ± 1.2 µA/cm2 with a
PDte and Rte of
17.5 ± 0.6 mV and 568 ± 24
· cm2, respectively.
Addition of amiloride to these
F-HBE reduced Isc by 29.6 ± 1.5 µA/cm2 to
a new plateau value of 5.1 ± 0.2 µA/cm2
(n = 116). Thus amiloride reduces
Isc an average of 50% in wt HBE, whereas it
reduces Isc by 87% in
F-HBE,
demonstrating a significant Na+ hyperabsorption in our
F-HBE cultures (P < 0.0001). Our data on wt
CFTR-expressing HBE are presented first, and data on
F-HBE are
presented in later sections.
Effect of Forskolin on Ion Transport Across HBE
The effect of forskolin (10 µM), subsequent to amiloride, on ion transport across wt HBE is shown for one monolayer in Fig. 1A. Forskolin induced an initial peak in Isc followed by a sustained plateau (see also Figs.2 and 6). In 119 monolayers forskolin increased Isc from an amiloride-inhibited plateau of 16.0 ± 0.7 to 30.0 ± 1.4 µA/cm2 followed by a decline to a stable plateau of 24.4 ± 1.1 µA/cm2. As shown in Fig. 1A, the Na+-K+-2Cl
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Our results suggest that a portion of the amiloride-insensitive
Isc is due to Cl secretion.
However, the Isc does not approach zero in the
presence of the combination of amiloride plus bumetanide, suggesting an additional ongoing active transport process. Smith and Welsh
(47) previously demonstrated that canine trachea was
capable of secreting HCO3
in response to
cAMP-mediated agonists and also demonstrated that primary cultures of
human airway respond to forskolin in Cl
-free solutions,
suggesting a HCO3
secretory process. Also, we
recently demonstrated that Calu-3 cells secrete HCO3
in response to elevated cAMP (16). Thus, in an initial
attempt to determine whether a portion of the amiloride-insensitive
Isc observed may be due to
HCO3
secretion, we utilized a combination of the
carbonic anhydrase inhibitor acetazolamide (100 µM) and the
Na+/H+ exchange inhibitor EIPA (5 µM). As
shown in Fig. 1A, acetazolamide plus EIPA reduced
Isc an additional 3.6 ± 0.4 µA/cm2 (n = 4), suggesting this basal
Isc may be due to HCO3
secretion. The magnitude of this inhibition is similar to what was
reported by Smith and Welsh (47) using acetazolamide (1 mM) and serosal amiloride (1 mM) in canine trachea.
To further evaluate the possibility that forskolin is stimulating
HCO3 secretion across wt HBE, we performed ion
substitution experiments in which Cl
or both
Cl
and HCO3
were removed from both the
mucosal and serosal solutions (see METHODS). As shown in
Fig. 1B, in the absence of Cl
, forskolin
stimulated a bumetanide-insensitive increase in
Isc that was partially inhibited by
acetazolamide. In 11 experiments forskolin increased
Isc an average of 6.3 ± 1.3 µA/cm2 in the absence of Cl
. By comparison,
in the absence of both Cl
and HCO3
,
forskolin increased Isc by only 0.9 ± 0.4 µA/cm2 (n = 6). These experiments
demonstrate that forskolin stimulates a transepithelial current
response that is dependent on HCO3
in the bathing solution.
We recently demonstrated that the human airway cell line Calu-3
secretes HCO3 by a Na+-dependent
mechanism in response to forskolin and that this could be inhibited by
serosal DNDS (16). High concentrations of DNDS (1-3
mM) have been shown to inhibit the
Na+-HCO3
cotransporter (4,
56), suggesting that this cotransporter was responsible
for HCO3
entry across the serosal membrane of Calu-3
cells [ribonuclease protection assays confirm expression of a
Na+- HCO3
cotransporter (NBC) in Calu-3
cells as well as HBE (Gangopadhyay NN and Bridges RJ, unpublished
observations)]. We therefore determined whether DNDS would similarly
inhibit forskolin-mediated anion transport across wt HBE. As shown in
Fig. 1C, in the absence of mucosal and serosal
Cl
, serosal DNDS (3 mM) partially inhibited the
forskolin-induced Isc, and this was further
reduced by the addition of acetazolamide. In five monolayers, forskolin
increased Isc from 3.4 ± 0.3 to 7.4 ± 1.0 µA/cm2, and this was reduced to 5.4 ± 0.8 and 3.7 ± 0.5 µA/cm2 by DNDS and acetazolamide,
respectively. These results suggest that a DNDS-sensitive
Na+- HCO3
cotransporter is partially
responsible for serosal HCO3
entry in HBE, a
mechanism similar to that which we described in Calu-3 cells
(16).
Although it is likely that CFTR represents the apical membrane
Cl channel activated by forskolin, the basolateral
membrane K+ channels involved in maintaining the driving
force for Cl
secretion have received little attention.
Therefore, we evaluated the effect of several known K+
channel blockers on the forskolin-stimulated
Isc. We previously demonstrated that CTX
inhibits Ca2+- but not cAMP-dependent Cl
secretion across T84 cells (15). Similarly, in wt HBE
cells, CTX (50 nM) had no effect on forskolin-stimulated
Cl
secretion (Fig. 2).
Similar results were obtained in six additional experiments. Lohrmann
et al. (34) first described the chromanol, 293B,
as a highly selective inhibitor of the basolateral membrane cAMP-dependent K+ channel. We determined the effect of 293B
(100 µM) on the forskolin-stimulated Isc in wt
HBE (Fig. 2). In 12 monolayers, forskolin increased Isc from 17.1 ± 2.8 µA/cm2
to a sustained value of 23.4 ± 3.4 µA/cm2, and this
was reduced by 67% to 19.2 ± 2.7 µA/cm2 by 293B
(P < 0.001). These results demonstrate that, similar to colonic epithelia (15, 34), human airway
expresses a 293B-sensitive K+ conductance.
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Although it is clear that 293B inhibits a significant portion (67%) of
the cAMP-mediated Isc, a large 293B-independent
Isc remains (Fig. 2). Our results above (Fig. 1)
suggest that a portion of this current is due to Cl
secretion based on its bumetanide sensitivity and requirement for
Cl
. Thus we determined whether other nonselective
K+ channel blockers would further inhibit this basal
Cl
secretion. Subsequent to 293B, addition of
Ba2+ (5 mM) reduced Isc to a
sustained value of 8.5 ± 1.5 µA/cm2
(n = 10; Fig. 2), with bumetanide further reducing
Isc to 3.7 ± 0.5 µA/cm2
(n = 10). Subsequent to 293B, quinine further reduced
Isc from 9.8 ± 0.6 to 4.3 ± 0.8 µA/cm2 (n = 4). Finally, clofilium (100 µM) inhibited both the forskolin-dependent and -independent current,
reducing Isc from 42.0 ± 12.5 to 4.7 ± 1.2 µA/cm2 (n = 4). These results
suggest that there is a Ba2+-, clofilium-, and
quinine-sensitive basolateral K+ conductance that underlies
the bumetanide-sensitive Cl
secretion induced by
amiloride and forskolin.
Effect of the CFTR Openers NS004, 8-MOP, and Genistein on
Cl Secretion Across wt HBE
NS004.
Gribkoff et al. (23) characterized NS004 as the first
known opener of both wt and F508 CFTR in the Xenopus
oocyte heterologous expression system. Subsequently, we demonstrated
that, although NS004 increased apical membrane Cl
conductance in the T84 cell line, it failed to induce a
Cl
secretory response (13). Also, NS004
failed to stimulate Cl
secretion in primary cultures of
MTE (13). In contrast to these results, NS004 (10 µM)
stimulated a sustained, bumetanide-sensitive increase in
Isc across wt HBE, subsequent to amiloride (Fig.
3A). This concentration of
NS004 was chosen based on both the Ks (see below) for NS004 as well as our previous studies, demonstrating that
higher concentrations of NS004 disrupted epithelial integrity in T84
cells (13). In 18 experiments, NS004 increased
Isc by 10.8 ± 1.7 µA/cm2,
from 13.4 ± 0.9 to 24.2 ± 2.1 µA/cm2. In
eight of these experiments, the subsequent addition of bumetanide reduced Isc to 10.4 ± 0.6 µA/cm2. In nine additional monolayers, a complete
concentration-response curve for NS004 was generated. These data were
fitted to a Michaelis-Menten function having an apparent
Ks of 1.2 ± 0.3 µM (Fig. 3B).
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Psoralens.
We previously demonstrated that the psoralens increase apical membrane
Cl conductance and stimulate Cl
secretion
across both T84 cells and MTE (14). Similar to our results
with NS004, stimulation of transepithelial Cl
secretion
required the addition of a K+ channel agonist such as
1-EBIO or carbachol (14). Based on blocker pharmacology,
we proposed that the psoralens stimulated Cl
secretion
via an activation of CFTR (14). We evaluated the effect of
8-MOP in wt HBE following inhibition of basal Na+
absorption with amiloride. The maximal effective concentration of 8-MOP
for increasing apical GCl in T84 cells was 30 µM. To directly compare the effects of NS004 and 8-MOP, we chose to
utilize 10 µM 8-MOP for our HBE studies. As shown in Fig.
4, 8-MOP induced a sustained,
bumetanide-sensitive increase in Cl
secretion across wt
HBE. In five monolayers, 8-MOP increased Isc
from 10.0 ± 2.3 to 20.0 ± 0.4 µA/cm2.
Addition of bumetanide reduced this current to 9.7 ± 1.4 µA/cm2. These results confirm that, following
hyperpolarization of the apical membrane with amiloride,
Cl
channel agonists are capable of stimulating
Cl
secretion across wt HBE. In addition, we determined
the effect of 8-MOP on Isc in the absence of
amiloride. 8-MOP had no significant effect on Na+ transport
(see Effect of NS004, 8-MOP, 1-EBIO, and Genistein on
Na+ Absorption Across
F-HBE), suggesting any
increase in Isc was due to anion secretion.
Similar to NS004, 8-MOP induced a small, albeit significant increase in
Isc of 3.7 ± 1.3 µA/cm2
(n = 5, P < 0.05) in the absence of
amiloride.
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Genistein.
Genistein has been shown to stimulate Cl secretion across
both T84 (28) and Calu-3 (26) cells, and more
recently has been demonstrated to directly activate both wt and
F508
CFTR in excised patches (25, 54). We
therefore determined whether genistein would stimulate transepithelial
Cl
secretion across wt CFTR-expressing HBE. As shown in
Fig. 5A, subsequent to
amiloride, genistein (50 µM) induced an initial peak increase in
Isc followed by a sustained,
bumetanide-sensitive plateau. This concentration of genistein was
chosen based on previous studies (27, 45) as
well as on the Ks determined in our own studies
(see below). In 17 monolayers, genistein induced an initial peak increase in Isc from 11.0 ± 1.3 to
18.9 ± 1.2 µA/cm2 followed by a sustained plateau
at 16.6 ± 1.3 µA/cm2. The addition of bumetanide in
nine of these experiments reduced Isc to
5.4 ± 0.7 µA/cm2. In an additional six monolayers a
complete concentration-response relationship was generated for
genistein and the data fitted to a Michaelis-Menten function having an
apparent Ks of 1.9 ± 0.4 µM (Fig.
5B). We were unable to evaluate the effects of genistein on
anion secretion in the absence of amiloride as we have demonstrated that genistein stimulates a significant increase in Na+
absorption across HBE (12) (see Effect of NS004,
8-MOP, 1-EBIO, and Genistein on Na+
Absorption Across
F-HBE). Thus
changes in Isc could not be reliably attributed
to either cation absorption or anion secretion.
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Effect of the Ca2+-Dependent
K+ Channel Opener 1-EBIO on
Cl Secretion Across wt HBE
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Our results above suggest that forskolin, as well as the
Cl channel openers, genistein and NS004, stimulate
HCO3
secretion across wt HBE, in addition to their
effects on Cl
secretion. Because 1-EBIO activates both
CFTR and KCa (15), we determined whether
1-EBIO would similarly stimulate Isc in a
Cl
-independent manner, suggestive of
HCO3
transport. In the absence of mucosal and serosal
Cl
, 1-EBIO (1 mM) increased Isc
from 4.4 ± 0.3 to 8.1 ± 1.1 µA/cm2
(n = 3). Addition of serosal DNDS (3 mM) and
acetazolamide (100 µM) reduced Isc to 6.3 ± 0.3 and 5.1 ± 0.2 µA/cm2, respectively,
suggesting that 1-EBIO stimulates HCO3
secretion
across wt HBE.
Effect of Mucosal UTP on Cl Secretion Across HBE
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In the T84 colonic cell line the response to the
Ca2+-mediated agonist, carbachol, is potentiated by both
Cl channel activators, including forskolin
(17), NS004 and 1-EBIO (13), and psoralens
(14), as well as compounds that impinge on second
messenger pathways such as wortmannin (phosphatidylinositol 3-kinase) (52) and arachidonyl trifluoromethyl
ketone (AACOCF3) [phospholipase A2
(PLA2)] (2, 11). Indeed, we
demonstrated that NS004 (13) and the psoralens
(14) not only potentiated the initial increase in
Isc across T84 cells but also induced a
sustained phase to the carbachol response. Potentiation of either the
transient or sustained phase of a Ca2+-mediated response in
HBE would be expected to be of therapeutic benefit. Therefore, we
evaluated the effect of these compounds on the
Isc response to mucosal UTP across wt HBE. In
contrast to previous results from T84 cells, the effect of UTP on
Cl
secretion across wt HBE was unchanged in the presence
of NS004 (10 µM;
Isc = 10.9 ± 2.7 µA/cm2; n = 4), 1-EBIO (1 mM;
Isc = 5.0 ± 0.2 µA/cm2; n = 9), AACOCF3 (100 µM;
Isc = 8.9 ± 1.6 µA/cm2; n = 6) or wortmannin (100 nM,
Isc = 10.1 ± 2.3 µA/cm2; n = 7). These results suggest
that the pharmacological potentiation of Ca2+-mediated
agonists observed in other model Cl
secretory systems
cannot be readily extrapolated to human bronchial epithelia.
The response to mucosal UTP (100 µM) in the presence of forskolin is
shown for one experiment in Fig.
8A. Subsequent to forskolin, addition of mucosal UTP induced a further increase in
Isc that was followed by a decline to below the
initial forskolin plateau level. In a total of 30 monolayers, mucosal
UTP increased Isc by only 7.0 ± 1.2 µA/cm2, with the plateau current level being 4.9 ± 0.6 µA/cm2 below the sustained forskolin-induced current
level. These results demonstrate that the response to mucosal UTP is
not potentiated by forskolin. Indeed, the mucosal UTP-induced increase
in Isc in the presence of forskolin is smaller
than that observed in the absence of forskolin (P < 0.001). Also, the net effect of UTP is a decrease in total outward
current. The net decrease in outward current observed could be due to
an inhibition of Cl secretion or a stimulation of
K+ secretion. Indeed, we have previously shown that mucosal
UTP inhibits the Ba2+- and quinine-sensitive basolateral
GK likely responsible for maintaining the
driving force for Cl
secretion in the presence of
forskolin (12) (Fig. 2). To determine whether the decrease
in Isc observed was specific for UTP, we determined the effect of the Ca2+-ATPase inhibitor,
thapsigargin. Similar to mucosal UTP, following establishment of a
sustained forskolin response, thapsigargin (1 µM) induced an initial
small increase in Isc
(
Isc = 3.9 ± 0.8 µA/cm2; n = 10) followed by a decline in
current to 4.3 ± 0.9 µA/cm2 below the sustained
forskolin current level. This result indicates the decrease in current
observed is independent of receptor-mediated effects.
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We next determined whether the initial increase in
Isc induced by UTP is due to the activation of a
CTX-sensitive basolateral membrane K+ channel. As shown in
Fig. 8B, CTX (50 nM) completely inhibited the initial
increase in Isc induced by mucosal UTP, whereas
the apparent inhibitory phase was unaffected. In seven monolayers, the
initial increase induced by UTP in the presence of CTX averaged 0.4 ± 0.4 µA/cm2, whereas in seven paired
experiments, carried out on the same day on monolayers from the same
patient, UTP induced a significantly greater increase in
Isc, averaging 12.9 ± 3.9 µA/cm2 (P < 0.001). These results
demonstrate that the initial increase in Cl secretion
induced by UTP is due to the activation of a basolateral membrane,
CTX-sensitive K+ channel. Prior microelectrode studies
confirm that mucosal ATP induces a large decrease in basolateral
membrane resistance with no change in the electromotive force of the
basolateral membrane, consistent with activation of both basolateral
GK and GCl
(6). In this case, inhibition of GK
with CTX would result in a depolarization of the basolateral membrane
toward the Cl
reversal potential
(ECl) such that
Isc is decreased.
Ca2+-Dependent
K+ Secretion Across wt and F-508
HBE
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The observation that glibenclamide inhibited the
Ca2+-dependent K+ secretory response led us to
determine whether increased Ca2+ would similarly stimulate
K+ secretion across F508 HBE in a
glibenclamide-dependent fashion. As shown in Fig. 9B,
thapsigargin stimulated K+ secretion across
F508 HBE
that was sensitive to block by mucosal glibenclamide (100 µM). The
subsequent addition of mucosal Ba2+ (5 mM) further
inhibited Isc. In six experiments, thapsigargin increased Isc from 56 ± 12 to 235 ± 28 µA/cm2. Addition of glibenclamide and Ba2+
reduced Isc to 56 ± 6 and 18 ± 1 µA/cm2, respectively. These results demonstrate that
Ca2+-dependent agonists similarly activate an apical
membrane, glibenclamide-sensitive GK in
F508
HBE. Also, these results suggest that the magnitude of the
UTP-dependent Cl
secretory response observed may be
underestimated, as any simultaneous UTP-dependent K+
secretory response would produce a current of opposite polarity.
Effect of Pharmacological Modulators on Cl Secretion
Across
F-HBE
1-EBIO.
Before evaluating the effects of 1-EBIO on transepithelial
Cl secretion across
F-HBE, we determined whether the
basolateral membrane K+ conductance in
F-HBE would
respond to pharmacological (1-EBIO) modulation. The mucosal membrane
was permeabilized with nystatin and a serosa-to-mucosa K+
gradient established across the epithelium to measure serosal membrane
K+ currents (IK; see
METHODS). After nystatin permeabilization, 1-EBIO (1 mM)
increased IK from 11.3 ± 1.2 to 31.0 ± 2.8 µA/cm2 (n = 8), and this was inhibited
by CTX (50 nM; 13.8 ± 1.2 µA/cm2), consistent with
activation of a basolateral membrane Ca2+-dependent
K+ conductance (10, 15). This
response to 1-EBIO was similar in magnitude to that induced by
mucosal UTP (
IK = 31.2 ± 6.9 µA/cm2, n = 9).
8-MOP.
We next evaluated the effect of 8-MOP on ion transport across F-HBE,
subsequent to inhibition of Na+ absorption with amiloride.
Similar to 1-EBIO, 8-MOP failed to significantly increase
Isc, subsequent to amiloride. In four
monolayers, amiloride reduced Isc from 44.1 ± 5.1 to 1.8 ± 0.2 µA/cm2, with the subsequent
addition of 8-MOP (10 µM) increasing Isc to
only 2.2 ± 0.4 µA/cm2.
NS004.
Because NS004 stimulates Cl secretion across
HBE-expressing wt CFTR (Fig. 4), we determined whether this proposed
CFTR activator would stimulate Cl
secretion in
F-HBE.
As shown in Fig. 10A,
subsequent to amiloride, NS004 (10 µM) had little effect on
Isc. The subsequent addition of forskolin (10 µM) induced an increase in Isc, which was
sensitive to bumetanide. In 13 experiments NS004 failed to
significantly increase Isc (0.4 ± 0.3 µA/cm2), whereas the subsequent addition of forskolin
increased Isc (2.3 ± 0.3 µA/cm2). The effect of NS004 on
Isc was also evaluated subsequent to forskolin
addition (Fig. 10B). Forskolin increased
Isc an average of 1.4 ± 0.3 µA/cm2. In contrast to NS004 alone, addition of NS004
subsequent to forskolin induced a significantly greater,
bumetanide-sensitive, increase in Isc (2.1 ± 0.3 µA/cm2, n = 21, P < 0.001). These results suggest that forskolin and NS004 act in a
synergistic fashion to stimulate Cl
secretion across
F-HBE (Fig. 10C).
|
Genistein.
The effect of genistein (50 µM) on Cl secretion across
F-HBE is shown in Fig. 11.
Subsequent to amiloride, genistein induced a small,
bumetanide-sensitive increase in Isc. In
contrast to our results with NS004, addition of forskolin caused no
further increase in Isc following genistein. In
11 experiments, genistein increased Isc an
average of 2.5 ± 0.5 µA/cm2 (P < 0.01), with the subsequent addition of forskolin increasing Isc by only an additional 0.4 ± 0.5 µA/cm2. In an additional nine experiments carried out on
monolayers from the same culture, the order of forskolin and genistein
addition were reversed. In these monolayers forskolin increased
Isc by 0.5 ± 0.2 µA/cm2,
with genistein further increasing Isc by only
0.7 ± 0.5 µA/cm2.
|
Effect of Pharmacological Activators on F-HBE Cultured at 26°C
|
Effect of 1-EBIO and Forskolin on F508/2789
+5G
A HBE
Effect of NS004, 8-MOP, 1-EBIO, and Genistein on
Na+ Absorption Across F-HBE
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When comparing the effects of pharmacological modulators of
transepithelial ion transport between wt and F508 CFTR-expressing human airway, it is important to consider the source of the material. Although all of our wt CFTR-expressing HBE were from transplant tissue,
only one was from a bronchiectasis patient chronically colonized with
Pseudomonas aeruginosa, similar to our CF
cultures. Thus it could be argued that the milieu of the wt CFTR- vs.
F508 CFTR-expressing HBE, before culture, may impact the subsequent results. This is unlikely, however, because our monolayers were studied
3-5 wk following acquisition of the bronchial material, having
been cultured in two separate growth media. A second consideration is
that the cells cultured from these two patient populations are distinct
and therefore respond differently. However, both our wt CFTR and
F508 CFTR-expressing HBE were Na+ absorptive in the
basal state, consistent with a surface cell phenotype. In addition, our
CF cultures displayed both the hallmark hyperabsorption of
Na+ and diminished Cl
secretory responses
associated with in vivo measurements from CF patients (32,
33). Recently, both Engelhardt and colleagues (20, 61) and Dupuit et al. (18)
have shown that, after reconstitution (in xenografts) of epithelial
cells derived from diseased nasal or bronchial epithelium, the
distribution of airway cell phenotypes is not different than normal
airway. This suggests that a progenitor cell population is selected
with in vitro culture and that expansion and differentiation of this
population normalizes the morphological differences typically present
in the diseased lung. Finally, we have performed scanning electron
microscopy (SEM) and immunostaining for markers of cell differentiation
in airway cultures derived from both CF and non-CF human airway. By
SEM, the majority of cells in all filters examined had features of
differentiated airway cells, namely abundant microvilli and cilia, and
apical expression of the membrane mucin MUC1 (data not shown).
Collectively, these data suggest that the differences in ion transport
between the CF and non-CF airway cell filters do not reflect the
inflammatory milieu of the underlying disease or differences in
epithelial cell phenotype but rather are associated with the CF genotype.
Pharmacological Modulation of Cl Secretion
It has been proposed that pharmacological intervention designed to
increase the trafficking of F508 CFTR to the apical membrane would
be therapeutically beneficial. One means of testing this strategy is to
culture
F508 CFTR-expressing cells at 26°C (9). We
demonstrate that after incubation of
F-HBE at 26°C for 24 h,
the Cl
secretory response to NS004, genistein, and 1-EBIO
are all potentiated. These results further demonstrate that
pharmacological agents are capable of activating
F508 CFTR in human
airway epithelium.
An important question is, How much CFTR must be expressed at the apical
membrane to pharmacologically elevate anion secretion? Although we have
not quantified the levels of CFTR in the apical membrane of our
cultures, our studies on the CFTR splice site mutation 2789 +5GA
shed some light on this question. Highsmith et al. (24)
demonstrated that airway cells homozygous for the splice site
mutation 2789 +5G
A express ~4% wt CFTR mRNA, whereas the
remainder of the CFTR is stopped prematurely during translation. In cells heterozygous for the 2789 +5G
A mutation
(the other allele being
F508), 1-EBIO induced a sustained
Cl
secretory response, whereas in homozygous
F508
airway cells 1-EBIO had no measurable effect. Similarly, subsequent to
forskolin, 1-EBIO further increased Isc. These
results suggest that as little as 2% of wt CFTR mRNA is required to
generate sufficient protein to be pharmacologically manipulated.
CFTR is Rate Limiting in Both wt and F508 HBE
Our results suggest that a basolateral membrane
GK is constitutively active, thereby providing
the driving force necessary for Cl secretion in response
to the CFTR openers, NS004 and 8-MOP. We speculate this conductance is
responsible for maintaining the driving force for Na+ entry
across the apical membrane in the nonstimulated state. Addition of
amiloride has been shown to stimulate Cl
secretion across
human airway epithelia, due to a hyperpolarization of both apical
(Va) and basolateral membranes with a
concomitant increase in apical membrane fractional resistance
(58). These data indicate that the basolateral membrane is
not downregulated in parallel with the apical Na+
conductance. Thus this basolateral GK maintains
the electrochemical driving force for Cl
exit in the
presence of amiloride. These data also highlight the critical need to
evaluate potential CFTR openers in the appropriate context, since, in
the absence of a full complement of apical and basolateral
conductances, the electrochemical driving force acting on
Cl
may be inappropriate for identifying Cl
channel modulators.
HCO3 Secretion Across Human Airway
K+ Channels Involved in Ion Transport Across HBE
As is well known, the maintenance of both Na+ absorption and ClSimilar to our previous findings on T84 cells (15), the
forskolin-stimulated Cl secretory response was partially
inhibited by the cAMP-dependent K+ channel blocker, 293B
(Fig. 2) (34). Although it was originally proposed that
293B inhibited the min-K channel (IsK)
(49), more recent evidence suggests that the molecular
target of 293B is actually KvLQT1 (35).
Indeed, KvLQT1 has been shown to be expressed in both T84
cells and HBE (8) (unpublished observations), consistent with our blocker profile. In contrast to these results, MacVinish et
al. (36) reported that the cAMP response in murine nasal epithelia was insensitive to 293B. However, we have observed a 293B-inhibitable current in response to forskolin in primary cultures of MTE (Devor DC, unpublished observations). These results suggest that
murine nasal epithelia may not be an adequate model for human bronchial
epithelia with regard to K+ channel expression. Also, we
observed no effect of NS004 on Cl
secretion across MTE
(13), further suggesting that murine airway is an
inadequate model for predicting human airway ion transport. Indeed, the
observation that murine airway epithelia are unaffected in CFTR
knockout mice supports this supposition.
In addition to basolateral membrane K+ conductances, we demonstrate a significant K+ conductance in the apical membrane as well. This conductance was activated by increasing cellular Ca2+ but not cAMP, similar to what has recently been reported by Clarke et al. (7). Whereas our results do not distinguish between secretion and absorption, Clarke et al. (7) have demonstrated that the electrochemical driving force acting on K+ favors secretion across the apical membrane. Thus, whereas CFTR has been proposed to interact with ROMK at the apical membrane of kidney epithelia, thereby conferring glibenclamide sensitivity (40), we demonstrate that airway apical membrane K+ conductance expression and blocker pharmacology are independent of wt CFTR expression (Fig. 9).
Pharmacological Modulation of Na+ Absorption
CF is characterized not only by a diminished ClWe previously demonstrated that genistein stimulates Na+
absorption across HBE (12). Our present findings confirm
this observation (Fig. 13), suggesting that genistein may have the
unwanted effect of further increasing Na+ absorption across
CF airway in vivo. The mechanism by which this stimulation occurs
remains unclear, however. Genistein has been shown to directly activate
CFTR in excised patch-clamp recordings (25,
54). Thus one possibility is that genistein modulates Na+ transport via an interaction with CFTR, as CFTR itself
has been shown to negatively modulate ENaC activity (48).
However, the CFTR opener NS004 has no effect on Na+
transport, which argues against this possibility. A second possibility is that genistein activates a basolateral GK,
thereby increasing the driving force for Na+ absorption.
Indeed, direct activation of basolateral KCa by 1-EBIO or
UTP (12, 37) stimulates Na+
absorption. We have found that genistein stimulates Cl
secretion across T84 monolayers in a 293B-sensitive manner (unpublished observations), suggesting that genistein not only activates CFTR but
also basolateral GK. Thus we speculate that
genistein is activating a basolateral GK to
stimulate Na+ absorption across HBE. It should be noted
that the magnitude of the increase in Isc cannot
be accounted for by Cl
secretion, as genistein has only
modest effects on Cl
transport in
F-HBE even in the
presence of amiloride, where there would be an improved electrochemical
driving force.
In summary, we demonstrate that both CFTR (NS004, 8-MOP, genistein) and
KCa (1-EBIO) openers stimulate a sustained Cl
secretory response across HBE. In addition, all of these agonists also
stimulate HCO3
secretion, further suggesting that
CFTR is the exit pathway for HCO3
across the apical
membrane. Importantly, we demonstrate that NS004 and genistein are
capable of stimulating Cl
secretion across
F-HBE,
suggesting that some
F508 CFTR escapes the degradative pathway to be
expressed at the apical membrane, consistent with the results of Dupuit
et al. (18, 19). Indeed, a mutation expected
to produce only 2% of wt CFTR mRNA (2789 +5 G
A) results in
expression of sufficient CFTR in the apical membrane to produce a
pharmacological increase in anion secretion.
Implications for Development of New Therapies for Airway Diseases
Our results suggest pharmacological activators of CFTR or GK may prove clinically useful in several settings. Recent data suggest that impaired ClMucociliary clearance is impaired in several other airway diseases,
including chronic bronchitis and bronchiectasis (3, 53). Studies have suggested that -adrenergic agonists
and UTP improve mucociliary clearance in these diseases
(39, 42, 59), presumably via
increases in mucus secretion, ciliary beat frequency, and/or
Cl
secretion. Because
-agonists and methylxanthines
are bronchodilators, it is difficult to determine the contribution of
fluid secretion to their favorable clinical effects. However, as
demonstrated in this report, benzimidizalones, psoralens, and genistein
augment Cl
secretion in human airway epithelia and may
therefore be useful compounds for treatment of chronic bronchitis,
bronchiectasis, and other diseases in which impaired mucociliary
clearance contributes to airway obstruction. In support of this, recent
data in normal volunteers indicate that chlorzoxazone, a skeletal
muscle relaxant that is structurally related to 1-EBIO, and genistein,
each stimulate Cl
secretion in human nasal epithelium
(26, 46). Safety and dose-response studies to
determine the effect of these compounds on mucociliary clearance and
airways obstruction will be necessary to determine their utility as
adjunctive therapies for patients with chronic bronchitis and bronchiectasis.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the excellent secretarial skills of Michele Dobransky, the technical assistance of Cheng Zhang Shi in both tissue culture and Ussing chamber experiments, and Drs. Jan Manzetti and Robert Keenan of the University of Pittsburgh Medical Center lung transplant program for assistance in obtaining human lung tissue.
![]() |
FOOTNOTES |
---|
This work was supported by Cystic Fibrosis Foundation Grants DEVOR96P0 and Q933, by a CF Research Development Program Center grant, and by National Institute of Diabetes and Digestive and Kidney Diseases Grant P50-DK-56490.
Address for reprint requests and other correspondence: D. C. Devor, Dept. of Cell Biology and Physiology, S312 BST 3500 Terrace St., Univ. of Pittsburgh, Pittsburgh, PA 15261 (E-mail: dd2+{at}pitt.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. §1734 solely to indicate this fact.
Received 11 August 1999; accepted in final form 29 February 2000.
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