1 Department of Physiology, Previous studies demonstrated that ACh-induced
liquid secretion by porcine bronchi is driven by active
Cl
cystic fibrosis; cystic fibrosis transmembrane conductance
regulator; bronchi; epithelium; exocrine glands
THE CYSTIC FIBROSIS transmembrane conductance regulator
protein (CFTR) is a cAMP-regulated anion channel (1). In human, bovine,
and ferret lungs, this protein is found in both surface and glandular
epithelia of the airways, although the magnitude of expression is
greatest in the serous cells of the airway submucosal glands (4, 11,
19). In persons afflicted with cystic fibrosis (CF), the CFTR is
mutated, greatly reducing the ability of the airway epithelium to
secrete Cl Recent studies demonstrated that ACh-induced liquid secretion across
porcine bronchial epithelium is driven by active transport of both
Cl In the present study, we hypothesized that CFTR is present in the
airway submucosal glands of pigs and plays a significant role in the
secretion of both Cl Airway excision.
Young pigs (10-15 kg) were obtained from a local vendor. Animals
were sedated with intramuscular injections of ketamine (80 mg) and
xylazine (4 mg) and euthanized with an intravenous overdose of
pentobarbital sodium. The chest was rapidly opened, and portions of
both lungs were quickly excised and placed in Krebs-Ringer bicarbonate
(KRB) at room temperature. Distal bronchi (external diameter 2-3
mm, length 25-35 mm) were dissected from the lung lobes. Airway
branches were ligated with sutures adjacent to the central airway
trunk. The isolated bronchi were then placed in fresh KRB at room
temperature and warmed slowly to 37°C (~0.2°C/min).
Measurement of liquid secretion.
After reaching physiological temperature, the bronchi were pretreated
with the appropriate inhibitor (see below) for 45 min to ensure that
complete channel or transporter blockade had occurred. During this
pretreatment period, the inhibitors bathed both luminal and adventitial
surfaces of the tissues. At the end of this pretreatment period, the
airways were removed from the incubation solutions, and the lumens were
cleared of all liquid and mucus. The tissues were quickly cannulated
with polyethylene tubing and returned to their original incubation
solutions containing the inhibitors. ACh (10 µM) was then added to
the bath solutions to stimulate liquid secretion. This procedure was
followed to ensure that the airway lumen was free of liquid at the
start of the ACh exposure. After 2 h of exposure to ACh, the bronchi
were removed from their solutions and sectioned lengthwise. Mucus
liquid was collected from the lumen and placed in tared polyethylene
microcentrifuge tubes (0.5-ml capacity). Two different methods were
used to collect mucus liquid. One method utilized forceps and a small
glass rod to collect the mucus while residual liquid was collected with a pipette. The other method consisted of aspirating all luminal liquid
with polyethylene tubing attached to a 1-ml syringe. A previous study
demonstrated that these two techniques resulted in equivalent
volumetric recoveries (22). Samples were weighed with a Mettler H20
balance and frozen for later analysis. Secretory rate was
calculated from total secretion volume and ACh incubation time and
normalized to calculated surface area. Luminal surface area (SA) was
calculated from airway length (L)
and outer diameter (D) with the use
of the following relationship
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
and
HCO
3 secretion. The present study
was undertaken to determine whether this process was localized to
submucosal glands and mediated by the cystic fibrosis transmembrane
conductance regulator (CFTR). When excised, cannulated, and treated
with ACh, porcine bronchi secreted 15.6 ± 0.6 µl · cm
2 · h
1.
Removal of the surface epithelium did not significantly affect the rate
of secretion, indicating that the source of the liquid was the
submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a
relatively nonselective
Cl
-channel blocker,
significantly reduced liquid secretion by 86%, whereas pretreatment
with DIDS, which inhibits a variety of
Cl
channels but not CFTR,
had no effect. When bronchi were pretreated with glibenclamide or
5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR),
the rate of ACh-induced liquid secretion was significantly reduced by
39 and 91%, respectively, compared with controls. Agents that blocked
liquid secretion also caused disproportionate reductions in
HCO
3 secretion. Polyclonal
antibodies to the CFTR bound preferentially to submucosal gland ducts
and the surface epithelium, suggesting that this channel was localized
to these sites. These data suggest that ACh-induced gland liquid
secretion by porcine bronchi is driven by active secretion of both
Cl
and
HCO
3 and is mediated by the CFTR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
. The myriad of
pulmonary symptoms that characterize this disease, such as mucus
plugging of airways, increased susceptibility to airway infection, and
bronchiectasis, are thought to result in some way from impairment of
transepithelial anion secretion. However, a definitive paradigm linking
reduced anion transport with development of CF lung pathology has not
been established. Indeed, the normal function of airway surface liquid
as well as the site of origin of this fluid remains controversial.
and
HCO
3 (22, 23). Because inhibitors
of Cl
and
HCO
3 secretion also cause mucins
to accumulate within the ducts of the submucosal glands, it has been
reasoned that these agents selectively blocked liquid but not mucus
secretion from submucosal glands (7, 8). Whereas similar obstruction of
airway gland ducts is one of the first signs of CF disease in newborns
(15), these findings suggest that uncoupling of liquid and mucus
secretion also occurs in this disease, possibly representing the
primary pulmonary lesion in CF. The mechanism of ACh-induced
Cl
and
HCO
3 secretion from pig airways
appears to be consistent with the model proposed by Smith and Welsh
(21), who suggested that both Cl
and
HCO
3 cross the apical
membrane of airway epithelial cells through CFTR. Although evidence for
CFTR involvement in anion secretion has been reported for primary
cultures and cell lines derived from airway gland cells (12-14),
to our knowledge, CFTR has not been definitively linked to
physiological liquid secretion by submucosal glands in intact airways.
and
HCO
3 by glandular epithelium. To
test this hypothesis, we determined the immunohistochemical
distribution of CFTR in pig airways and examined the liquid secretion
response of these airways to a series of potential anion-channel
blockers. Our results indicate that CFTR is present in pig airway
glands and that it supports not only glandular
Cl
and liquid secretion but
HCO
3 secretion as well.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
A
previous morphometric analysis determined that the cross-sectional
inner surface circumference of porcine small bronchi averages 68.2% of
the outer surface circumference (Trout, unpublished observations).
Liquid volume (in ml) was considered to be equivalent to wet weight (in g).
Inhibitor pretreatments.
To demonstrate the role of
Cl channels in ACh-induced
airway liquid secretion, the bronchi were pretreated for 45 min with one of several agents known to inhibit
Cl
-channel activity: 100 µM diphenylamine-2-carboxylate (DPC), 1 mM DIDS, saturated (~350
µM) glibenclamide, and 300 µM
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Glibenclamide and
NPPB are thought to exhibit selectivity for the CFTR (12, 20). DPC and
DIDS block a wide spectrum of
Cl
channels, although DIDS
reportedly does not inhibit the CFTR (2, 5, 10). For comparison, some
airways were pretreated with both 10 µM bumetanide and 100 µM
dimethylamiloride (DMA), a pretreatment combination that has been
previously shown to block ~90% of ACh-induced liquid secretion from
bronchi by selectively inhibiting
Cl
and
HCO
3 secretion, respectively (22).
Control airways in each group were pretreated with equal volumes of the drug vehicle.
Epithelium removal. In some bronchi, the surface epithelium was removed to assess the contribution of these cells to the volume of secreted liquid. A wooden ream was inserted into the airway lumen and rotated both clockwise and counterclockwise to strip off the surface epithelium. Microscopic examination confirmed that this method effectively removed the surface epithelium while leaving the smooth muscle and submucosal structures intact. Epithelium removal was performed after dissection of the airways and before warming of the tissues.
Bicarbonate analysis and flux.
Frozen samples of airway mucus liquid were thawed and assayed for
HCO3 with an Infinity
CO2 kit (Sigma Diagnostics, St.
Louis, MO). This assay is based on the enzymatic conversion of
HCO
3 to oxaloacetate in the
presence of phospho(enol)pyruvate and
phospho(enol)pyruvate decarboxylase. Malate dehydrogenase then catalyzes reduction of oxaloacetate to malate and the oxidation of
NADH to NAD. The reduction in absorbance at 380 nm is proportional to
the amount of HCO
3 in the sample.
Absorbance of the reaction product was measured with a Beckman DU65
spectrophotometer. Sample concentrations were determined by comparison
to known standards. Because the airway lumen was nominally free of
liquid at the start of the 2-h ACh incubation period, determination of
the net flux of HCO
3 during this
period could be made from the total volume of secreted liquid and the
HCO
3 concentration in this volume.
Immunohistochemical detection of CFTR.
Lung tissue was frozen in optimum cutting temperature (OCT) embedding
medium. Thin (5-10 µm) sections were prepared with a cryostat
and fixed at 4°C in acetone for 10 min. Sections were air-dried and
stored at 20°C until further use. At the time of the assay,
sections were rehydrated in PBS. Endogenous peroxidase activity was
blocked with 0.1% (vol/vol) phenylhydrazine-HCl. After saturation of
nonspecific protein binding sites with nonimmune goat serum (Vector
Laboratories; diluted 1:50 in PBS for 20 min at room temperature), the
specimens were incubated at room temperature with purified anti-CFTR
(rabbit, polyclonal) first nucleotide-binding domain (NBD1) antibody
(16) for 1 h. Preimmune serum at the same concentration was used as a
negative control. After a wash with PBS, goat anti-rabbit horseradish
peroxidase-labeled antibody was added at 1:200 dilution and incubated
for 40 min at room temperature. Specific immune reaction was revealed
with the use of 3-amino-9-ethylcarbazole as chromogen and 0.1%
H2O2
as substrate, dissolved in 0.1 M sodium acetate buffer, pH 5.2. Mayer's hematoxylin was used as nuclear counterstain.
Statistics. Where paired comparisons are indicated, data were analyzed with dependent t-tests. Unpaired data were compared with independent t-tests. Data are reported as means ± SE, with n referring to the number of airways (each from a different animal) in each group. P < 0.05 was considered significant. Values that fell more than two standard deviations from the mean within each group were considered statistical outliers and were omitted from analysis.
Solution composition and drugs. KRB contained 112 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.6 mM glucose in aqueous solution. The pH of KRB was maintained at 7.4 by continuous gassing of solutions with 5% CO2 in O2. PBS (pH 7.4) contained 8.0 mM Na2HPO4, 2.0 mM KH2PO4, 120 mM NaCl, and 2.7 mM KCl. To make stock solutions, inhibitors were dissolved in DMSO. All drugs and chemicals except for glibenclamide (RBI), DPC (N-phenylanthranilic acid; Fluka Chemika), OCT (Fisher Scientific), and NPPB (Calbiochem) were purchased from Sigma Chemical.
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RESULTS |
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Immunohistochemical distribution of
CFTR.
Peroxidase staining in the airway sections of pig bronchi shows that
the purified anti-CFTR (rabbit, polyclonal) NBD1 antibody localizes to
both the surface epithelium and the submucosal glands (Fig.
1A).
The most intense labeling appears to be within the ducts of the
submucosal glands. Tissue sections incubated with preimmune serum show
only nonspecific labeling with minimal signal (Fig.
1B).
|
Effect of epithelium removal.
In the presence of ACh, the bronchi secreted 15.6 ± 0.6 µl · cm2 · h
1
(n = 67) (Fig.
2). The secretion response to ACh in
denuded airways (13.4 ± 1.7 µl · cm
2 · h
1;
n = 17) was not significantly
different from that in epithelium-intact airways. The rate of liquid
flux
(JV)
was significantly inhibited by pretreatment with 10 µM bumetanide and
100 µM DMA [a drug combination previously shown to inhibit
Cl
,
HCO
3, and liquid secretion across
porcine bronchi (22)] in both epithelium-intact (2.1 ± 0.3 µl · cm
2 · h
1;
n = 12) and epithelium-denuded airways
(3.2 ± 0.6 µl · cm
2 · h
1;
n = 8) (Fig. 2). The effect of
epithelium removal was also insignificant in inhibitor-treated tissues.
These results indicate that most of the liquid secreted by bronchi in
response to ACh arises from submucosal glands.
|
Effect of channel blockers.
Similar to previous studies (22), pretreatment with the combination of
10 µM bumetanide and 100 µM DMA, which selectively target the
Na+-K+-2Cl
cotransporter and the Na+/H+ exchanger,
respectively, inhibited 86.5% of the ACh-induced liquid secretion from
porcine bronchi (Table 1). This action has
been attributed to respective inhibition of transepithelial
Cl
and
HCO
3 secretion by these agents
(22). Comparable inhibition of ACh-induced liquid secretion was
observed when airways were pretreated with 100 µM DPC or 300 µM
NPPB. Glibenclamide (~350 µM) pretreatment significantly blocked
38.5% of the secretion response, whereas 1 mM DIDS pretreatment had no
effect.
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DISCUSSION |
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We made the following observations during this study. First, antibodies
to the CFTR densely stained the epithelial cells of the airway
submucosal glands of porcine bronchi. Second, porcine bronchi when
treated with ACh secreted liquid at similar rates in both the presence
and absence of the surface epithelium. Third, Cl-channel blockers that
are known to inhibit the CFTR were effective blockers of ACh-induced
liquid secretion and disproportionately reduced
HCO
3 secretion. These findings
reasonably support the hypothesis that CFTR is present in submucosal
glands of pig airways and participates in the secretion of
Cl
,
HCO
3, and liquid from these structures.
Earlier studies by Inglis et al. (9) of the bioelectric properties of
porcine bronchi provided evidence that ACh stimulates both
Cl and
HCO
3 secretion from these tissues.
Subsequent studies demonstrated that inhibitors of
Cl
and
HCO
3 secretion not only blocked
the liquid secretion response to ACh (22, 23) but caused mucins to
accumulate within the ducts of the submucosal glands (9, 16). The
authors of these studies reasoned that this secreted liquid likely
originated from submucosal glands, although they could not rule out
significant contributions from nonglandular airway regions. The results
of the present study confirm these earlier notions by showing that similar quantities of liquid are secreted into the lumen of airways in
the presence or absence of the surface epithelium. Inhibitors of
Cl
and
HCO
3 secretion substantially
reduce this secretion response even when the surface epithelium is
removed. Consequently, we conclude that the secreted liquid emanates
largely from submucosal epithelium (i.e., glands) and is driven by
active anion secretion.
Unfortunately, potent, highly selective inhibitors of the CFTR are
presently unavailable. Therefore, demonstration of CFTR involvement in
complex tissues must be inferred from the pattern of responses to
several semiselective
Cl-channel inhibitors. DPC
blocks the CFTR, Ca2+-activated
Cl
channels, outwardly
rectifying Cl
channels, and
a variety of other Cl
channels (2, 10). In the present study, DPC was an effective inhibitor
of the ACh-induced liquid secretion response, consistent with
Cl
-channel involvement.
DIDS inhibits Ca2+-activated
Cl
channels, outwardly
rectifying Cl
channels, and
many of the other DPC-sensitive
Cl
channels but not the
CFTR (2, 5, 10, 24). Consequently, blockade of ACh-induced secretion by
DPC but not by DIDS implies a functional role for the CFTR. Both NPPB
and glibenclamide are also known to inhibit the CFTR (12, 20). In the
present study, NPPB was as effective as DPC in blocking liquid
secretion, but glibenclamide inhibited only ~40% of the response.
The reason why incomplete inhibition of the
JV occurred with
glibenclamide is unclear. This was probably not a dose effect, since
maximum (i.e., saturating) concentrations of glibenclamide were used. It is more likely that this effect reflects the lower intrinsic efficacy of glibenclamide compared with the other CFTR blockers. Vandorpe et al. (24) showed in inner medullary collecting duct cells
that the order of effectiveness for absolute reductions in the product
of CFTR-channel number and open probability was NPPB > DPC > glibenclamide, with DIDS having no effect. This same order of inhibitor
effectiveness is seen in the present study for reductions in both
liquid volume and HCO
3 secretion.
Blockade of liquid secretion with DPC and NPPB is unlikely to be the
result of overt toxicity because we observed that the ACh secretion
response was fully restored after washout of these inhibitors. We
conclude that the responses to this group of inhibitors are most
consistent with participation of the CFTR in the liquid secretion
response to ACh.
Cl-channel inhibitors that
would be expected to block the CFTR also appear to inhibit
HCO
3 secretion. DPC and NPPB
reduced HCO
3 secretion more than
predicted based on reductions in liquid volume secretion, whereas DIDS
had no effect. Even glibenclamide, which reduced liquid secretion by
only 40%, caused a fractionally greater and significant inhibition of
HCO
3 flux. This inhibitory action
of these agents on HCO
3 secretion
clearly targets glandular epithelium and not surface epithelium, since
reductions in HCO
3 flux were also
observed in bumetanide- and DMA-pretreated airways with the epithelium
removed. These data suggest that
HCO
3 secretion from glands is
mediated through the CFTR. Involvement of the CFTR in the secretion of
HCO
3 in airways is supported by
reports that the CFTR is permeable to this anion (5, 17) and that it
mediates HCO
3 secretion by
duodenal epithelia (3, 6).
In addition to providing functional evidence for CFTR involvement in gland secretion, we showed by immunohistochemistry that CFTR is present in gland epithelium of pigs. Although the distribution is shown for polyclonal antibodies to CFTR, we saw a similar expression pattern for monoclonal antibodies as well (data not shown). These findings support those of others reporting high levels of CFTR expression in airway glands of humans, cows, and ferrets (4, 11, 19) and imply a functional role for CFTR in anion transport by these structures.
Cl concentrations and
fluxes were not reported in the present study because the quantity of
Cl
present in the smallest
(<5 µl) sample volumes was too low for accurate determination with
conventional potentiometric
Cl
titration. Trout et al.
(22) showed in previous studies that ~70% of ACh-induced liquid
secretion from pig bronchi is driven by bumetanide-sensitive
Cl
secretion, whereas
HCO
3 secretion accounts for only
~10-20%. Because DPC and NPPB block ~85-90% of the
liquid secretion response,
Cl
secretion must have been
nearly abolished by these agents. Partial inhibition of
Cl
secretion must have
occurred even with glibenclamide because the observed 40% inhibition
of the JV exceeds
the estimated maximum contribution of
HCO
3 secretion by at least
twofold. Therefore, we conclude that these agents exert a large
fraction of their inhibitory effects on liquid volume secretion through inhibition of active transepithelial
Cl
secretion, which
accounts for most of the active anion secretion response to ACh by this tissue.
In previous studies, we observed that bumetanide and DMA, inhibitors of
epithelial Cl and
HCO
3 secretion, respectively,
induced changes in porcine bronchi that resembled those expected to
occur in CF disease. First, we saw abolishment of ACh-induced
electrogenic anion secretion (9), an expected consequence of defective
CFTR expression. Second, we found that these inhibitors greatly reduced ACh-induced airway liquid secretion (22), confirming that this process
is dependent on active anion secretion. Again, this would be the
anticipated result of defective CFTR expression as occurs in CF. Third,
we observed that the bronchial mucus that is secreted after the
Cl
and
HCO
3 secretion inhibitors is less
hydrated and more rigid and exhibits less recoil than control mucus
(23). This finding supports the commonly held yet controversial notion that the CF condition leads to secretion of relatively dehydrated mucus
with altered rheological properties. Fourth, we found that bumetanide
and DMA together cause mucin accumulation and an obstruction of
submucosal gland ducts of airways (7, 8) that closely resembles the
gland duct obstruction that occurs very early in CF disease
(15). Bumetanide and DMA produce their inhibitory effects
on Cl
and
HCO
3 secretion by selectively
targeting Na+-K+-2Cl
cotransport and
Na+/H+
exchange (22). If
Cl
and
HCO
3 both flow across the apical
membrane of glandular epithelial cells through CFTR, we expect that
equivalent inhibition of liquid secretion would occur with blockers of
this channel. The results of the present paper confirm this hypothesis, with DPC and NPPB producing equivalent if not greater inhibition of
liquid secretion than bumetanide+DMA. We expect that these same
Cl
-channel blockers would
be useful for modeling the acute pathophysiology of CF disease in
surrogate species such as the pig, in which airway gland expression
resembles that of humans.
If blockade of the CFTR by DPC or NPPB acutely mimics the CF condition,
the pH of airway surface liquid in CF patients might be relatively more
acidic than normal. In the present study, control airway surface liquid
contains 20-25 mM HCO3, whereas liquid from NPPB-treated airways contains only 8.1 ± 3.2 mM
HCO
3. With the assumption that all
solutions were equilibrated with 5%
CO2, the Henderson-Hasselback
relationship predicts that the pH of airway surface liquid would be
~7.33-7.45 in control airways and as low as 6.95 in
inhibitor-treated airways. It is possible that this approximately
threefold higher H+ concentration
is relevant to the development of CF lung pathology. However, findings
that the pH of cholinergically induced airway surface liquid is
normally ~7.0 in ferrets (18), a species that exhibits no unusual
propensity toward airway disease, argues against this notion.
Because we expected the surface epithelium to provide a significant barrier to passive liquid movement, we were surprised to observe that the rate of liquid flux into the lumen of epithelium-denuded and epithelium-intact airways was very similar. Cannulated airways were submerged vertically in Krebs buffer; therefore, interstitial fluid pressure ranged from 0 cmH2O to as much as +5 cmH2O relative to the air-filled lumen in some tissues. This hydrostatic pressure gradient should have favored liquid flow into the airway lumen. From our data, it is clear that little passive liquid flow into the lumen occurred when the epithelium was removed. We speculate that passive liquid movement through the interstitial space was restricted by the gellike properties of the interstitial matrix and perhaps by the uniform layer of smooth muscle cells that lies within the airway wall.
In summary, we show that airway submucosal glands are the principal
site of ACh-induced liquid secretion from porcine airways. We also
provide evidence that CFTR is present in submucosal glands and supports
not only Cl and liquid
secretion but HCO
3 secretion as well. By demonstrating a physiological function for the CFTR in glandular transepithelial anion and liquid secretion, we are hopeful that these findings provide a crucial link toward our understanding of
the pulmonary pathogenesis of CF.
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ACKNOWLEDGEMENTS |
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This work was funded by the National Institutes of Health Grants HL-48622 and SCOR DK-53090 and Cystic Fibrosis Foundation RDP Grant R464.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: S. T. Ballard, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688 (E-mail: sballard{at}usamail.usouthal.edu).
Received 2 April 1999; accepted in final form 2 June 1999.
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