1 Department of Environmental Health Sciences and 3 Department of Physiology and Biophysics and Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 2 Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506
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ABSTRACT |
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Cystic fibrosis (CF), an inherited
disease characterized by defective epithelial Cl
transport, damages lungs via chronic inflammation and oxidative stress.
Glutathione, a major antioxidant in the epithelial lung lining fluid,
is decreased in the apical fluid of CF airway epithelia due to reduced
glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ.
Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether
restoration of chloride transport would also restore glutathione
secretion. We found that a Cl
channel-forming peptide
(N-K4-M2GlyR) and a K+ channel activator
(chlorzoxazone) increased Cl
secretion, measured as
bumetanide-sensitive short-circuit current, and glutathione efflux,
measured by high-performance liquid chromatography, in a human CF
airway epithelial cell line (CFT1). Addition of the peptide alone
increased glutathione secretion (181 ± 8% of the control value),
whereas chlorzoxazone alone did not significantly affect glutathione
efflux; however, chlorzoxazone potentiated the effect of the peptide on
glutathione release (359 ± 16% of the control value). These
studies demonstrate that glutathione efflux is associated with apical
chloride secretion, not with the CF transmembrane conductance regulator
per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.
chlorzoxazone; channel-forming peptide; glutathione transport; antioxidant
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INTRODUCTION |
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CYSTIC
FIBROSIS (CF) is caused by mutations in the CF transmembrane
conductance regulator (CFTR) gene, which encodes a cAMP-dependent Cl channel located primarily in the apical membrane of
epithelial cells. Lung dysfunction, manifested as mucus accumulation,
chronic infection, and chronic inflammation, is a major cause of death in CF. Chronic inflammation results in severe oxidative stress in CF
patients (29).
In CF lungs, invading neutrophils produce superoxide and hydrogen peroxide and release myeloperoxidase, which catalyzes formation of hypochlorous acid. This chronic oxidative stress is exacerbated by decreased antioxidant capacity in both the airway lining fluid and plasma of CF patients (3, 10). The fluid covering the lung epithelium normally contains a high concentration of the antioxidant glutathione (GSH) (4, 27). GSH content is decreased in the plasma and bronchoalveolar lavage fluid from CF patients and the apical fluid from CF airway epithelial cells (10, 24). Furthermore, Gao et al. (10) have recently shown that decreased apical GSH content in CF is caused by defective GSH efflux. Because GSH is particularly effective in reducing hypochlorous acid (9), the major protein-damaging agent in CF sputum (29), the defective secretion of GSH has a potentially profound effect on the course of the disease.
Although it is now known that CFTR deficiency causes decreased GSH export (10), little else is known regarding GSH export from airway epithelial cells. In general, the relationship between anion conductance and the GSH transporter system(s) is poorly understood. In isolated rat hepatocytes, GSH transport is membrane potential dependent (8). GSH efflux in the rat liver is also carrier mediated, possibly by the multidrug resistance protein (MRP) (19, 21). More recently, CFTR itself has been suggested to be permeable to GSH by the patch-clamp technique (16). Nonetheless, significant residual GSH secretion is present in CF airway cells, suggesting that CFTR may regulate transport rather than carry GSH.
Although transfection of normal CFTR remains a therapeutic goal,
treatment of symptoms and the use of antibiotics are considered the
predominant therapeutic strategies in CF. Recently, novel pharmacological approaches have been used to restore Cl
transport in CF, including activation of defective CFTR
(25), activation of alternative non-CFTR Cl
channels (20), and incorporation of an artificial
Cl
channel-forming peptide into the apical membrane of
epithelial cells (22, 32).
Reddy et al. (22) and Wallace et al.
(32) have described synthetic peptides that form a
Cl-permeable pathway, bypassing the low abundance of CFTR
Cl
channels in Madin-Darby canine kidney monolayers.
M2GlyR is a synthetic 23-amino acid peptide, PARVGLGITTVLTMTTQSSGSRA,
derived from the second transmembrane segment of the
-subunit of the glycine receptor, a glycine-gated Cl
channel. This
peptide self-assembles into a presumed pentamer, forming
anion-selective channels in phospholipid bilayers (22). Four lysine residues can be added to either the NH2
terminus (N-K4-M2GlyR) or the COOH terminus
(C-K4-M2GlyR) of M2GlyR to increase its solubility and
activity (28). These peptides induce transepithelial
Cl
and fluid transport in Madin-Darby canine kidney
cells, human colonic cells (T84), and CF airway epithelial cells
(IB3-1) (17, 32). A scrambled peptide containing a random
sequence of the same amino acid composition as M2GlyR plus four lysine
residues shows no Cl
secretory activity
(32). The flux of Cl
through the artificial
Cl
channel is upregulated by increasing basolateral
K+ transport through Ca2+-dependent
K+ channels in T84 cells (31).
Antioxidant therapy has also been proposed to combat chronic
inflammation in the CF lung (24). GSH is a major
antioxidant, and its content is reduced in the CF apical fluid due to
defective GSH secretion (10). Therefore, the mechanism of
GSH efflux and the possibility of stimulating GSH efflux in CF patients
by pharmacological intervention by increasing Cl
permeability were investigated in the present work. A synthetic Cl
channel-forming peptide was used to bypass
CFTR-dependent Cl
transport. This allowed us to determine
whether the GSH efflux is dependent on Cl
transport or
CFTR. We conclude that GSH efflux is effectively increased by
restoration of Cl
secretion across CF airway epithelial
cells and that GSH release is associated with Cl
transport but not with CFTR per se. This suggests a potential new
therapy in which correcting a Cl
secretion defect may
prevent oxidative stress and improve the quality of life of CF patients.
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METHODS |
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Cell lines and cell culture.
CFT1 cells are immortalized human tracheal epithelial cells from a
homozygous F508 CFTR CF patient (33). CFT1-LCFSN cells are the same CFT1 clone stably transduced with normal CFTR
(18). The cells were grown in serum-free Ham's F-12
medium supplemented with seven additives: 5 µg/ml of insulin, 3.7 µg/ml of endothelial cell growth supplement, 25 ng/ml of epidermal
growth factor, 5 µg/ml of transferrin, 1 × 10
6 M
hydrocortisone, 3 × 10
8 M triiodothyronine, and 10 ng/ml of cholera toxin. The cells were cultured in collagen-coated
flasks for growth and collagen-coated permeable inserts (Becton
Dickinson, Bedford, MA) for treatments at 37°C in 5%
CO2. Before being seeded on the permeable membrane, the
cells were passaged once in Ham's F-12 medium without cholera toxin.
Short-circuit current measurements.
Confluent cell monolayers were mounted in Ussing chambers (Jim's
Instrument, Iowa City, IA) and bathed with symmetrical Ringer solution
containing 2.5 mM K2HPO4, 2.0 mM
CaCl2, 1.2 mM MgSO4, 5 mM
D-glucose, 5 mM sodium acetate, 6 mM L-alanine,
1 mM sodium citrate, 115 mM NaCl, 4 mM sodium lactate, 0.5 mM
n-butyric acid, 20 mM NaHCO3, and 14.1 mM
raffinose (pH 7.4). The temperature was maintained at 37°C, and the
solutions in both compartments were equilibrated with 5%
CO2. Short-circuit current (Isc) was measured with a voltage clamp (Physiologic Instruments, San Diego, CA)
as previously described (30). Initially, 10 or 100 µM
amiloride was added to the apical solution to inhibit activity of the
epithelial sodium channel. Then, different concentrations of the
peptide N-K4-M2GlyR were applied to the apical side
followed 50-80 min later by the addition of 500 µM chlorzoxazone
to both sides. Chlorzoxazone is a basolateral K+ channel
activator with a structure and activity similar to
1-ethyl-2-benzimidazolinone (6, 26). Both drugs increase
the driving force for Cl secretion across the epithelia
(6, 26).
Intracellular and extracellular GSH measurements with HPLC.
Experiments were performed after the cell monolayers developed a
maximum transepithelial resistance (~7-9 days). To examine the
effect of the peptide on GSH secretion, the medium was replaced with
0.5 and 1.5 ml of Ringer solution to the apical and basolateral sides,
respectively. Acivicin (0.2 mg/ml) was added to the Ringer solution to inhibit extracellular GSH degradation by -glutamyl transpeptidase. The channel-forming peptide was added to the apical solution, and where indicated, 500 µM chlorzoxazone was added to both
sides. The cells were incubated for 4 h at 37°C in 5% CO2.
Statistical analysis. Data are expressed as means ± SE and were evaluated by one-way analysis of variance followed by Tukey's test. P < 0.05 was considered significant.
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RESULTS |
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Elevation of Isc in CF cells with a Cl
channel-forming peptide and a K+ channel activator.
We investigated whether Cl
secretion was improved in CF
cells by treatment with a Cl
channel-forming peptide
(N-K4-M2GlyR) (28). CFT1 cells formed resistive monolayers (211 ± 24
· cm2)
with a basal Isc of 6.0 ± 0.4 µA/cm2 in ~7-9 days (n = 30 experiments). The effects of N-K4-M2GlyR and other agents
on Isc are shown in Fig.
1. The addition of amiloride, an
inhibitor of the epithelial Na+ channel, to the apical
bathing solution abolished the basal Isc. Subsequent addition of the peptide N-K4-M2GlyR caused
Isc to increase slightly over 40-60 min.
However, the addition of 500 µM chlorzoxazone, a K+
channel activator, ~50-80 min after the peptide treatment evoked a rapid elevation in Isc (P
0.001). Chlorzoxazone alone increased Isc
slightly. The marked increase in Isc caused by
N-K4-M2GlyR plus chlorzoxazone was abolished by the
addition of 300 µM bumetanide, a blocker of the Na-K-2Cl
cotransporter, to both sides of the monolayer. This suggests that the
Isc is mediated by Cl
secretion.
Dose-response data for N-K4-M2GlyR plus 500 µM
chlorzoxazone are shown in Fig. 2.
Another Cl
channel-forming peptide,
C-K4-M2GlyR (0.5 mM), plus chlorzoxazone (0.5 mM) also
increased Isc but was less potent than
N-K4-M2GlyR plus chlorzoxazone (3.17 ± 0.50 vs.
10.50 ± 1.98 µA/cm2). This difference in activity
is consistent with previous observations (2). These data
indicate that the synthetic channel-forming peptide plus chlorzoxazone
elicits a Cl
-secretory response across cultured CF airway
cells.
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Restoration of GSH secretion in CF cells by a Cl
channel-forming peptide and a K+ channel activator.
Next, we investigated whether GSH release was related to the addition
of agents that restore Cl
secretion across CF cells. The
rate of efflux was assayed by measuring GSH in the apical solution
after a 4-h incubation period (Fig. 3).
The addition of 0.5 mM N-K4-M2GlyR alone increased GSH secretion (181 ± 8% of the control value; P < 0.05), whereas the addition of chlorzoxazone alone did not
significantly affect GSH efflux in CFT1 cells. However, chlorzoxazone
potentiated the effect of the peptide, causing, for instance, an
increase in GSH release of 359 ± 16% of the control value with a
concentration of N-K4-M2GlyR of 0.5 mM (P < 0.001). We tested a second peptide, C-K4-M2GlyR, for
activity and observed that it alone also enhanced GSH secretion but
less potently than N-K4-M2GlyR alone (1 mM each peptide;
175 ± 9 vs. 252 ± 10% of control values). Thus
C-K4-M2GlyR has decreased efficacy compared with
N-K4-M2GlyR in both Isc and GSH
efflux assays. The addition of 300 µM 1-ethyl-2-benzimidazolinone,
another K+ channel activator, also amplified the effect of
N-K4-M2GlyR (0.5 mM) on GSH efflux (286 ± 1 vs.
181 ± 8% of control values). As shown in Fig.
4, intracellular GSH content was reduced
as the concentration of N-K4-M2GlyR increased with the
addition of chlorzoxazone. Therefore, our data suggest that treatment
with N-K4-M2GlyR in combination with chlorzoxazone corrects
the defect of GSH efflux in CFT1 cells by stimulation of
Cl
secretion.
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Blockade of GSH efflux in CF and non-CF cells by Cl
channel inhibitors.
To further determine the relationship between GSH efflux and
Cl
transport, CFTR-sufficient cells (CFT1-LCFSN) and CF
cells (CFT1) were treated with the Cl
channel inhibitors
glibenclamide and DNDS, and GSH efflux was measured (Table
1). Glibenclamide significantly
reduced GSH efflux in CFT1-LCFSN cells (58 ± 3% of the control
value; P < 0.001), whereas DNDS had no effect. In
contrast, neither glibenclamide nor DNDS altered GSH efflux from CFT1
cells. The ability of glibenclamide to selectively reduce GSH efflux
from cells expressing wild-type CFTR provides further evidence that GSH
and Cl
secretion are closely linked. These data also
indicate that airway epithelial cells possess both Cl
secretion-dependent (i.e., glibenclamide-sensitive) and -independent (glibenclamide-insensitive) GSH efflux pathways.
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DISCUSSION |
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CF is characterized by oxidative stress and chronic inflammation
in the respiratory tract. Decreased levels of GSH in the apical fluid
from CF airway cells (10) and CF patients
(24) may increase the susceptibility of the airway to
oxidative damage during chronic inflammation. Therefore, the
possibility of restoring the apical GSH content to protect the
integrity of the lung epithelium in CF patients is of great interest.
We explored whether administration of pharmacological agents causing an
increase in Cl secretion would also enhance GSH secretion.
One approach to stimulate Cl secretion is the use of
artificial Cl
channels (28, 32). In the
present study, the N-K4-M2GlyR peptide enhanced
Cl
secretion when added to CFT1 cells together with
chlorzoxazone. This is in agreement with earlier findings by Broughman
et al. (2). Chlorzoxazone itself had a small effect on
Isc across CFT1 cells. An earlier study
(26) showed that chlorzoxazone stimulates transepithelial
Cl
transport by activation of basolateral K+
channels in normal airway epithelium but not in the nasal epithelium of
CF patients, suggesting that apical Cl
-permeable pathways
are necessary for the chlorzoxazone effect. Amiloride was used to
confirm that the peptide-mediated current was carried by
Cl
instead of Na+. Although it is
demonstrated here that N-K4-M2GlyR peptide formed anion
channels permeable to Cl
, it does not rule out the
possibility of the permeability of other anions, including
HCO
permeability
suggests that the basolateral membrane is the rate-limiting factor for
Cl
secretion across CFT1 cells. This is supported by an
earlier study (6) that showed that activation of
basolateral K+ channels is necessary for the
Cl
-secretory response in T-84 cells.
N-K4-M2GlyR alone increased GSH efflux in CFT1 cells, which
was further enhanced by the addition of chlorzoxazone (Fig. 3). The
effect of the peptide on GSH release is dose dependent. The dose-response curves of apical GSH content and
Isc follow similar patterns but do not correlate
perfectly. N-K4-M2GlyR alone did not significantly affect
Isc but caused GSH release. Because
Isc indicates transepithelial ion transport,
whereas GSH is released from inside the cells to the apical space,
perhaps the peptide alone may slightly increase the Cl
permeability across the apical membrane. However, because the basolateral membrane is the rate-limiting factor, changes in
Isc are not significant without the addition of
chlorzoxazone to increase K+ transport across the
basolateral membrane. On the other hand, a small increase in
Cl
permeability across the apical membrane might be
sufficient to cause some elevation in GSH secretion. When chlorzoxazone
was added, it provided a driving force, causing more Cl
to be secreted across the apical membrane and a significantly enhanced
GSH release. N-K4-M2GlyR (0.5 mM) plus chlorzoxazone increased GSH efflux ~3.6-fold, which is comparable to the apical GSH
content in CFTR-sufficient cells (CFT1-LCFSN). This indicates that by
increasing Cl
secretion, the defect of GSH secretion
could be overcome in CF cells. As the concentration of the peptide
increased, more intracellular GSH was released to the extracellular
space (Fig. 4).
Although the identity of the GSH transporter is not presently known,
the studies here provide some insight into the relationship between
Cl transport and GSH efflux. The inability of
N-K4-M2GlyR to release GSH in liposomes suggests that
N-K4-M2GlyR itself does not function as a GSH transporter.
GSH has a net negative charge, and transport was shown to be dependent
on the membrane potential in hepatocytes and renal plasma membranes
(8, 11, 14). However, our results presented here cannot be
explained solely by changes in potential difference. Chlorzoxazone
increases the basolateral K+ permeability in CFT1 cells
(data not shown), which causes the membrane potential to hyperpolarize.
On the other hand, Cl
is initially accumulated in CFT1
cells via the bumetanide-sensitive Na-K-2Cl cotransporter in the
absence of CFTR (Fig. 1). The addition of N-K4-M2GlyR
allows more Cl
to exit, thereby depolarizing the membrane
potential. However, chlorzoxazone amplified the effect of
N-K4-M2GlyR on GSH efflux. Because chlorzoxazone and
N-K4-M2GlyR have opposing effects, it is unlikely that GSH
releases solely by changing the membrane potential. Rather, our data
suggest that GSH efflux is generally related to Cl
efflux. Specifically, apical anion permeability is essential (i.e.,
N-K4-M2GlyR or CFTR), and the membrane potential plays an
important although secondary role. Changes in intracellular potential
alone are not sufficient to affect GSH efflux because chlorzoxazone
itself did not affect GSH release. However, in the presence of an
apical Cl
-secretory pathway (N-K4-M2GlyR),
changes in membrane potential (chlorzoxazone) altered the GSH release
as shown in Fig. 3. Although the intracellular potential may be quickly
restored after perturbation, it is possible that
N-K4-M2GlyR in combination with chlorzoxazone may have
changed the intracellular potential substantially and maintained it at
that level under our experimental conditions.
An earlier study (16) suggested that CFTR conducts GSH
based on an increased current as measured with the patch-clamp
technique with the addition of GSH; however, no definitive evidence has shown that the CFTR protein itself transports GSH. The activity of CFTR
itself is believed to mediate the activity of other ion channels and
enzymes (1, 12) and to influence fetal development (5, 13) as well; however, as with the transport of GSH,
the underlying mechanisms of these regulatory events are a relatively new but intense area of investigation. With the use of the
Cl channel-forming peptide N-K4-M2GlyR, the
CFTR-dependent Cl
-permeable pathway was bypassed in the
present study. Therefore, our results suggest that GSH release is
associated with Cl
permeability rather than with
the CFTR protein per se. Further studies are necessary to characterize
the specific protein(s) that transport GSH.
Glibenclamide, an inhibitor of CFTR activity, inhibited GSH efflux in
CFTR-sufficient cells (CFT1-LCFSN) but not in CFTR-deficient cells
(CFT1). CFT1-LCFSN cells treated with glibenclamide are similar to CFT1
cells because they are CFTR defective. This is in agreement with the
earlier findings by Gao et al. (10) that GSH release is
decreased in CFTR-defective cells. This provides further evidence that
GSH and Cl secretion are closely linked. DNDS is a
Cl
channel inhibitor that is a very poor CFTR blocker
under physiological conditions. The lack of effect of DNDS on GSH
release in CFT1-LCFSN cells suggests that CFTR is the major
Cl
channel in these cells and that the non-CFTR-dependent
Cl
transport in this cell type is absent or very small.
The lack of effect of glibenclamide in CFT1 cells suggests that the
residual GSH secretion in these cells is CFTR independent. Because DNDS did not make any difference in GSH release from CFT1 cells, endogenous non-CFTR-dependent Cl
transport appeared insufficient to
affect GSH transport in CF cells. Taken together, these results
indicate that there are Cl
transport-dependent and
-independent pathways for GSH efflux in airway epithelial cells.
A diagram of predicted GSH efflux pathways based on the results
from the present study is shown in Fig.
5. In normal airway epithelial cells,
CFTR provides the major Cl-secretory pathway. Both
Cl
-dependent and -independent GSH transports are
functioning. MRP is a potential candidate for GSH transporter
(21, 34), although it is not clear whether MRP-mediated
GSH transport is Cl
dependent. The Nernst equation was
used to predict the direction of GSH movement:
Vapical
Vintracellular =
[zF/(RT)] × ln([GSH]apical/[GSH]intracellular), where Vapical and
Vintracellular are the apical and intracellular potentials, respectively; z is the valence; F is
the Faraday constant; T is the absolute temperature;
R is the gas constant; and [GSH]apical and
[GSH]intracellular are the apical and intracellular GSH
concentrations, respectively. For example, if the
[GSH]intracellular is 1,000 µM and the
[GSH]apical is 400 µM, the calculated potential
difference (Vapical
Vintracellular) will be
23.88 mV. Because the
Vintracellular is negative compared with the
Vapical, the measured
Vapical
Vintracellular is positive. Because the signs of
the measured and calculated potential differences are different, the
direction of the electrical potential force and the GSH concentration
gradient force are in the same direction. This means it favors GSH
secretion into the apical fluid.
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In CF airway epithelial cells, CFTR-dependent Cl
transport pathway is blocked. This inhibited Cl
transport-dependent GSH transport because CFTR is the major pathway for
Cl
transport. GSH is released to the apical fluid only by
a Cl
transport-independent system. Apical GSH
concentration is decreased. Although the GSH concentration gradient
force favors more GSH to be released to the apical fluid from CF cells
compared with normal cells, GSH cannot be secreted to the apical fluid
due to the defect in the Cl
transport-dependent GSH
transport system.
When the Cl channel-forming peptide
N-K4-M2GlyR is applied to the apical side, it forms another
Cl
-secretory pathway. This restores the function of the
Cl
transport-dependent GSH transport system. Addition of
the basolateral K+ channel activator chlorzoxazone
accelerates the K+ recycling, therefore providing a driving
force for Cl
secretion into the apical fluid. In
addition, chlorzoxazone also hyperpolarizes the cells by allowing more
K+ leakage to the basolateral fluid. Both the increase in
Cl
secretion and the hyperpolarization by chlorzoxazone
trigger more GSH secretion to the apical fluid. However, chlorzoxazone (hyperpolarization) alone is not sufficient to cause any GSH secretion in the absence of Cl
secretion.
Recently, the use of GSH aerosol has been suggested to suppress inflammatory cell-derived oxidants in the lungs of CF patients (23). Nonetheless, delivery of GSH to the interface between the neutrophils and the epithelium may be achieved most effectively by secretion directly through the epithelial surface. Our results provide a possible approach to increase apical GSH content where it is needed. The hydrophobic part of the peptide N-K4-M2GlyR makes it relatively easy to cross the thick mucus to reach the target airway epithelia. Restoration of GSH secretion by delivery of this peptide may lead to a potential clinical application for treatment against damage due to oxidative stress during chronic inflammation in CF patients.
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ACKNOWLEDGEMENTS |
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We thank Dr. James R. Yankaskas and Ronald Kim (University of North Carolina, Chapel Hill, NC) for providing the CFT1 and CFT1-LCFSN cell lines and Drs. Eric J. Sorscher, Julio Girón-Calle, and Kwang-Jin Kim for critical comments.
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FOOTNOTES |
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This work was supported by National Institute of Environmental Health Sciences Grant ES-05511; National Heart, Lung, and Blood Institute Grant HL-46943; National Institute of General Medical Sciences Grant GM-43617; National Institute of Diabetes and Digestive and Kidney Diseases Grant P30-DK-54781; Cystic Fibrosis Foundation Grant R464; and the Webb-Berger Foundation. Support was also provided in part by the Kansas Agricultural Experiment Station (KAES Manuscript 01-224-J).
Original submission in response to a special call for papers on "CFTR Trafficking and Signaling in Respiratory Epithelium."
Address for reprint requests and other correspondence: H. J. Forman, Dept. of Environmental Health Sciences, Univ. of Alabama at Birmingham, 1530 3rd Ave. S, RPHB 317, Birmingham, AL 35294-0022 (E-mail: hforman{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 6 November 2000; accepted in final form 30 November 2000.
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