1 Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688; and 2 Mucobiology Research Unit, Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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ABSTRACT |
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The combination of
both Cl and
secretion inhibitors causes an
accumulation of mucins within the submucosal gland ducts of
acetylcholine (ACh)-treated bronchi [S. K. Inglis, M. R. Corboz,
A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol.
Physiol. 16): L372-L377, 1997], suggesting indirectly that these agents block airway gland liquid secretion. The
present study tested the hypotheses that ACh-stimulated liquid secretion is driven by Cl
and
secretion and that inhibition
of this process leads to secretion of a dehydrated mucus with altered rheological properties. Excised distal bronchi from pigs were pretreated with either a combination of
Cl
and
secretion inhibitors (bumetanide, acetazolamide, dimethylamiloride, and
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) or the
dimethyl sulfoxide vehicle and were then treated with ACh to induce
secretion. The rate of mucus liquid secretion was substantially reduced
when the airways were pretreated with the anion secretion inhibitors.
Mucus liquid from inhibitor-pretreated airways contained almost
threefold more nonvolatile solids than the control liquid. Rheological
analysis revealed that mucus liquid from inhibitor-pretreated airways
expressed a significantly greater log
G* (rigidity factor), whereas tangent
(recoil factor) was significantly reduced. These results suggest
that 1) ACh-induced liquid secretion
in bronchi is driven by both
Cl
and
secretion and
2) inhibition of ACh-induced liquid
secretion results in the secretion of mucus with a reduced water
content and altered rheological properties.
cystic fibrosis; bicarbonate secretion; chloride secretion; mucus rheology; distal bronchi
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is a fatal inherited
disease that affects the exocrine function of many organs including the
lung, pancreas, small intestine, genitourinary tract, and sweat glands
(9). Patients typically succumb from an accumulation within the airways of an abnormally thick, purulent mucus and repeated bouts of severe, chronic pulmonary infection. CF is caused by mutations in the CF
transmembrane conductance regulator (CFTR) protein, which
functions in respiratory epithelia as a
Cl channel (1, 22).
Although currently the object of intense study and speculation, the
link between a defective CFTR and the production of abnormal airway
mucus in CF has not been conclusively demonstrated.
The CFTR may play several important roles in the maintenance of
cellular homeostasis. It has been variously suggested that CFTR
facilitates intracellular vesicular acidification (5), modulates
Na+-channel conductance (41),
mediates cellular efflux of ATP (37), and participates in the clearance
of inhaled bacteria (33). However, the function of CFTR that has
received the most attention in recent years relative to CF disease is
its role in adenosine 3',5'-cyclic monophosphate
(cAMP)-mediated Cl
secretion. Indeed, Cl
secretion is either present or inducible in airway epithelia (3, 8) and
serves as the likely driving force for transepithelial liquid secretion
(20). CFTR is also permeable to
(34) and could support active secretion of this anion as well (39).
Recent evidence suggests that the
Cl
and
secretion response to
acetylcholine in the airways is localized, at least in part, to the
bronchial submucosal glands, where it probably functions to drive
liquid secretion needed to flush glycoprotein secretions from the ducts (17). These observations are provocative because both CFTR and carbonic
anhydrase are localized to the serous cells of the airway submucosal
glands (11, 19, 40) and because gland obstruction and hypertrophy are
among the earliest pathological findings in CF disease (32, 38). These
observations support the notion that the uncoupling of liquid and mucin
secretions from the submucosal glands could be the central event in the
development of CF airway disease.
Mucus from the airways of CF patients differs in many respects from the mucus obtained from disease-free persons. CF mucus contains products of the omnipresent bacterial infections, including mucoid secretions, lipids, DNA, and actin, as well as proteases and other enzymes. Some of these substances exert significant effects on the physical properties of airway mucus that could substantially alter the efficiency of ciliary clearance (12, 13, 35). Consequently, it is very difficult to determine with certainty whether the physical differences between CF and normal mucus are due to a defective primary secretion product or the result of postsecretion modification by pathogenic and inflammatory processes. If anion and liquid secretions from airway submucosal glands are indeed defective in CF, the primary mucus secretion product could have a reduced water content compared with normal mucus, leading to altered rheological properties and a decreased ciliary or cough clearance.
Inglis et al. (17) recently reported that blockade of both
Cl and
secretion causes significant
mucin accumulation within the submucosal gland ducts of bronchi that were treated with acetylcholine (ACh), a glandular liquid and mucin
secretogogue. These findings were taken as indirect evidence that
liquid secretion from submucosal glands was driven by secretion of
Cl
and
. If this hypothesis is correct,
inhibitors of Cl
and
secretion uncouple liquid
secretion from mucin secretion and thereby create an ion and liquid
transport deficiency that resembles CF. The aims of the present study
were 1) to confirm that ACh-induced
liquid secretion in distal bronchi is blocked by inhibitors of
Cl
and
secretion and
2) to determine whether such an
intervention alters the physical properties of normal airway mucus.
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METHODS |
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Airway excision. Young pigs (10-15 kg, 7-8 wk old) of either sex were obtained from a local vendor. The animals were sedated with an intramuscular injection of ketamine (80 mg) and xylazine (4 mg) and euthanized with an intravenous overdose of pentobarbital sodium. Portions of either the right or left lung were quickly excised and placed in Krebs-Ringer bicarbonate solution (KRB) at room temperature. Distal bronchi (external diameter 2-3 mm, length 25-35 mm) were dissected from the surrounding parenchyma. Airway segments typically had 8-10 side branches. These branches were ligated with 6-0 sutures as close as possible to the central airway trunk. Isolated bronchi were then warmed slowly (~0.2°C/min) from room temperature to 37°C.
Collection of airway mucus liquid.
Bronchi were pretreated with a combination of four drugs to inhibit
Cl and
secretion: bumetanide (0.01 mM)
was used to inhibit
Na+-K+-2Cl
cotransport, acetazolamide (1 mM) was used to block carbonic anhydrase,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) was used to block
Cl
/
exchange, and dimethylamiloride (DMA; 0.1 mM) was used to inhibit
Na+/H+
exchange. This drug combination has been shown to block the
electrogenic anion secretion response to ACh and to cause mucin
accumulation within the gland ducts of ACh-treated tissues (16, 17).
Control tissues were treated with an equal volume of the dimethyl
sulfoxide (DMSO) vehicle. After an incubation period of 45 min, the
bronchi were removed from the KRB bath and cleared of all luminal
liquid and mucus. The bronchi were then cannulated with polyethylene tubing and returned to their respective solutions. To stimulate gland
secretion, 0.01 mM ACh was then added to the KRB bath, which contained
the drug pretreatments, and the tissues were incubated in the solutions
for an additional 2 h. When the incubation was complete, the bronchi
were removed from the cannulas and cut open lengthwise. Because the
mucus liquid was often too thick to be adequately sampled with
pipettes, this fluid was collected from the airway lumen with a 1- to
2-mm-diameter glass rod and forceps. This collection method was
possible because the non-Newtonian properties of mucus permit this
fluid to be picked up and manipulated as a semisolid. If present in the
cannulas, the mucus liquid was gently blown out of the tubes with
forced air. The mucus liquid was rapidly transferred to a tared piece
of aluminum foil and immediately weighed with either a Mettler H20
balance or a Cahn C-22 microbalance. Secretory rate was calculated from
the total secretory volume and time of incubation and was normalized to the estimated luminal surface area of the tissue. Luminal surface area
was calculated from the airway length and cross-sectional lumen
circumference. The latter value was assumed to be circular and was
estimated from the luminal diameter. In the present study, liquid
volume (in ml) was considered to be equivalent to wet weight (in g).
Measurement of nonvolatile mucus solids. After wet weight determination, the mucus liquid samples were dried overnight at 80°C. The dried samples were then removed from the oven, cooled to room temperature in a desiccator, and reweighed. Mucus dry weights could be reproducibly determined with a Mettler H20 balance when the liquid volumes exceeded 3 µl. However, reproducible measurements of the dry weights for 0.5- to 3.0-µl mucus samples required the use of a Cahn C-22 microbalance.
Measurement of mucus rheological properties. Mucus liquid was collected as described in Collection of airway mucus liquid and placed in tared 0.5-ml Eppendorf polypropylene centrifuge tubes. The tubes were immediately sealed, weighed to determine sample wet weight, and then quickly frozen. The samples were shipped on dry ice to the rheology laboratory, where they were allowed to warm to room temperature before they were tested.
The magnetic microrheometer technique was used to measure the viscosity
and elasticity of the mucus samples (26). Each mucus sample was placed
in a Plexiglas container and covered with light paraffin oil to
minimize evaporation. A 70- to 100-µm steel ball was inserted into
the mucus sample, and the container was positioned in the gap of a
toroidal electromagnet mounted on the stage of a projecting microscope.
The displacement of the ball, under the influence of a sinusoidally
oscillating magnetic field gradient, was measured. This ball acts as a
rheological probe because its movement is opposed by viscous and
elastic forces within the mucous gel fluid. The image of the steel ball
was projected onto a pair of photocells that provided an electrical
output in proportion to the displacement of the moving ball. The toroid
current (proportional to magnetic force) and photocell outputs were
transmitted to a digital oscilloscope connected to a plotter for
off-line processing. From the resulting measurements of driving force
and displacement, mucus viscoelasticity was determined at 1, 10, and
100 rad/s of applied frequency, and the results are expressed as log
G* (dyn/cm2) and tangent (loss
tangent), where G* is the vectorial sum of elasticity and viscosity, also considered as a rigidity factor,
and tangent
is a recoil factor, the viscosity-to-elasticity ratio.
Solution composition and drugs. The KRB contained (in mM) 112 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.6 glucose in an aqueous solution. Solution pH was maintained at 7.4 by continuous gassing with 5% CO2 in O2. All drugs and chemicals were purchased from Sigma.
Statistics. Within each experimental group, one control and one treated airway were obtained from each animal. Results were compared with either dependent or independent t-tests, with P < 0.05 as the level of significance. Data are reported as means ± SE.
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RESULTS |
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Mucus liquid secretion. Because the mucus liquid volumes from bronchi pretreated with transport inhibitors were very small (2.4-26 µl), evaporation of water from these samples during collection could have led to a significant error in the measurement of mucus hydration. To test this notion, mucus liquid samples of varying volumes were collected from ACh-treated bronchi. As shown in Fig. 1, the percentage of nonvolatile solids in the control mucus samples was consistent and not correlated to the sample volume (r2 = 0.0006). The solids content of mucus collected from bronchi pretreated with anion secretion inhibitors was consistently greater than that in control samples of comparable volume (Fig. 1).
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When bronchi were pretreated with the DMSO vehicle and then treated
with 0.01 mM ACh, mucus liquid was secreted at the rate of 0.24 ± 0.04 µl · cm2 · min
1
(n = 12 bronchi; Fig.
2). Nonvolatile mucus solids were 1.55 ± 0.01% (n = 10 bronchi) of the
total mucus liquid weight (Fig. 3), which
corresponds to 98.45% hydration. After pretreatment with anion
secretion inhibitors (0.01 mM bumetanide, 1.0 mM acetazolamide, 0.1 mM
DMA, and 1.0 mM DIDS), ACh-induced mucus liquid secretion was
significantly reduced to 0.02 ± 0.01 µl · cm
2 · min
1
(n = 12 bronchi; Fig. 2). Anion
secretion inhibition resulted in a significant threefold increase in
nonvolatile mucus solids to 4.31 ± 0.49%
(n = 10 bronchi; Fig. 3), equivalent
to 95.69% hydration.
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Mucus rheological properties. Four
control samples from bronchi pretreated with DMSO vehicle and six
samples from bronchi pretreated with the combination of anion secretion
inhibitors were analyzed for rheological (viscoelastic) properties by
magnetic microrheometry. Log G*
(rigidity factor) was determined at driving frequencies of 1, 10, and
100 rad/s. Tangent (recoil factor) was determined only at 1 and 10 rad/s because the rigidity and small size of some of the samples
resulted in insufficient tangent
data at 100 rad/s. Mean mucus
rigidity (log G*) was higher with
anion secretion inhibitor treatment at all three measurement frequencies (Table 1), and the
differences were statistically significant for the two lower
frequencies (1 and 10 rad/s). Tangent
(recoil) was significantly
lower for the anion secretion inhibitor group at 10 rad/s.
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DISCUSSION |
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These results confirm the previous suggestion by Inglis et al. (17)
that ACh-induced liquid secretion in distal bronchi is blocked by
inhibitors of Cl and
secretion. Additionally, we show
that inhibition of liquid secretion results in the production of a mucus with increased nonvolatile solids content, increased rigidity, and decreased recoil factor. These findings demonstrate that a deficiency in anion transport alone is sufficient to alter the physical
properties of airway mucus in a manner that predicts reduced airway
clearance.
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Much debate has centered on the etiology of the abnormal airway mucus
in CF patients. Some early studies using sputum failed to detect an
overall difference in mucus rheological properties compared with non-CF
lung disease (31) or normal animals (24), although abnormalities in
both the water and ion content of CF sputum have been well documented
(7, 42). Abnormalities in the rheological properties of CF mucus are
known to be related to the severity of infection (31, 35). The
interrelationships between mucus rheology and water and ion content are
made complex by the influence of the by-products of infection such as
leukocytic DNA and filamentous actin, which can add to the gel
cross-link density, and neutrophilic proteases, which are capable of
reducing network elements (10). Because virtually all CF patients
exhibit some level of airway infection, it is not possible to determine with certainty whether the primary glandular secretion product is
defective in these persons. The results of the present study indicate
that abolition of Cl and
secretion, which likely
corresponds to the situation in CF, is sufficient to decrease mucus
hydration, increase mucus rigidity, and decrease mucus recoil. Such
changes in the rheological properties should decrease mucus
transportability (28) and could possibly lead to mucus accumulation and
eventual obstruction of the bronchial airways. Furthermore, the
inability to remove inhaled pathogens from the airways could facilitate establishment of the chronic airway infections that typify CF.
The relationship between mucus viscoelasticity and clearance is complex. Clearability is impaired not only by increased viscosity and elasticity but also by an increased ratio of viscosity to elasticity (23). Furthermore, increases in solids content leading to correspondingly large increases in viscosity and elasticity, as seen in this study, may exert a relatively minor effect on clearability as long as the mucus retains its elastic character because it is the transfer of momentum from cilia to mucus that is critical in moving secretions by this mechanism. The dehydration of periciliary fluid may be much more consequential for mucus clearance because small changes in the depth of periciliary fluid could greatly alter the efficiency of mucus-cilia interaction. (6, 27). Although not directly testable in our model, the same processes that lead to the reduced water content of the mucus layer would likely also lead to dehydration of the periciliary fluid. The increased elasticity of the mucus would have a major retarding effect in clearance by airflow-related mechanisms such as cough and physiotherapy. Although not relevant to pig bronchi, similar dehydration mechanisms in CF airways would lead to mucus with a greatly reduced cough clearability (25).
The site of ACh-induced liquid secretion in distal bronchi cannot be precisely determined from the results of the present study. However, a significant fraction of the secreted liquid likely originates from the submucosal glands. The same combination of anion transport inhibitors used in the present study has been shown to cause occlusion of submucosal gland ducts in porcine bronchi, suggesting that gland liquid secretion is uncoupled from mucin secretion by these agents (17). Additionally, individual submucosal glands in the cat trachea have been shown to secrete liquid in response to atropine-sensitive vagal stimulation (44) and methacholine application (36). We cannot, however, discount the possibility that some fraction of the secretion response to ACh originates from the bronchial surface epithelium.
In this study, three drugs were used in combination to inhibit
secretion. Each agent targeted a
specific site that could potentially support
secretion. The model of Smith and
Welsh (39) for secretion of
from
airway epithelia suggests the presence of intracellular carbonic anhydrase and basolateral
Na+/H+
exchange. In the present study, these two sites were inhibited with
acetazolamide and DMA, respectively. Because of the possible existence
of basolateral
Cl
/
exchange in these tissues, DIDS was added to block
Cl
-dependent
secretion. Although we believe that these agents selectively target these sites, we cannot completely discount possible nonselective actions of these agents. For instance, DMA could also block conductive
Na+ channels in the apical
membrane of airway epithelial cells. However, this unintended effect
would not affect secretion but would block active absorption of
Na+ and liquid by surface
epithelial cells and should result in an increased rather than a
decreased net liquid production. DIDS is known to block non-CFTR
protein Cl
channels such as
Ca2+-activated, outwardly
rectifying, and volume-regulated
Cl
channels (2). However,
the combination of all three
secretion inhibitors, including DIDS, had no effect on the electrogenic anion secretion response to ACh in the absence of bumetanide (16), suggesting that the Cl
secretory pathway remains intact. It is unlikely that the combination of anion transport inhibitors is overtly toxic because mucus occlusion of gland ducts can be reversed by removal of the inhibitors and reapplication of ACh (Inglis, unpublished observations).
The combination of drugs used in the present study was intended to
block secretion, but the same
drug cocktail could potentially block
-dependent Cl
secretion as well.
However, recent findings suggest that
-dependent Cl
secretion plays no
significant role in ACh-induced liquid secretion in pig bronchi.
Preliminary studies show that of the three putative bicarbonate
secretion inhibitors, only DMA in combination with bumetanide is
required to block liquid secretion (4) and to cause mucin accumulation
within the submucosal gland ducts (15, 18). These data implicate
basolateral
Na+/H+
exchange as the critical site for inhibition and suggest that basolateral
Cl
/
exchange, which would be necessary for
-dependent
Cl
secretion to occur, does
not play a role in this process. Additional preliminary results show
that the concentration of
in the
secreted fluid as well as the total secreted
is reduced in tissues that have
been pretreated with DMA only (4; Ballard, unpublished
observations).
The results show that inhibitor pretreatment reduced mucus liquid
volume by 90% but caused only a threefold increase in mucus solids.
Consequently, the total quantity of mucus solids that were recovered
from the airway lumen was reduced approximately fivefold by inhibitor
pretreatment. The first and most plausible explanation for this
observation is that the missing mucus solids are trapped in the gland
ducts after Cl and
secretion inhibitor treatment.
Inglis et al. (17) confirmed histologically that mucins fill the ducts after inhibitor treatment. But because we cannot quantitatively measure
the total mass of mucus solids contained within the lumen of gland
ducts, this notion is difficult to prove beyond a doubt. A second
possibility is that we cannot collect all of the luminal mucus liquid
from the inhibitor-pretreated airways. This could contribute, in part,
to the missing mass, especially because the liquid volumes after
inhibitor pretreatment are so small. However, visual inspection of the
lumens of airways after mucus liquid collection revealed little
residual material, certainly not enough to account for a fivefold
difference in mucus solids. The third possibility is that the
Cl
and
transport inhibitors also inhibit mucin secretion. The presence of large amounts of mucin in gland ducts
after inhibitor pretreatment argues against this notion, although it is
possible that fractional inhibition of this process occurs.
For these results to have direct relevance to CF, it is presumed that
CFTR, a cAMP-activated Cl
channel, participates in active anion secretion induced by ACh. Because
ACh typically transduces its actions through
Ca2+, not cAMP, a role for CFTR in
this process could be questioned. Evidence is accumulating, however,
that CFTR is indeed involved in cholinergically stimulated anion and
liquid transport. Jiang et al. (21) and Yamaya et al. (45) showed that
CFTR most likely participates in cholinergically stimulated ion
secretion from cultures of airway gland cells and suggested that CFTR
is nearly maximally activated under baseline conditions. Additionally,
Hogan et al. (14) demonstrated that both cAMP-activated and
Ca2+-activated duodenal
secretion is mediated through
CFTR. The most likely explanation for these findings is that ACh
increases intracellular Ca2+
concentration, which opens basolateral
Ca2+-activated
K+ channels and hyperpolarizes the
cell membrane. This action increases the driving force for anion efflux
across the apical membrane, and, if CFTR is constitutively active,
anions could exit across apical cell membrane through this
channel.
Similar CF-related defects in exocrine function have been reported in
the pancreas. Pancreatic liquid secretion in CF patients is only
one-third that of non-CF patients, and the concentration of protein in
the pancreatic liquid is almost three times higher in CF patients than
control subjects (30). This reduction in pancreatic liquid secretion in
CF patients is concomittant with impaired
Cl and
secretion in this organ (29).
These observations in the pancreas mirror our own findings in the
bronchi where a block of Cl
and
transport reduces liquid
secretion, most likely from submucosal glands, resulting in a
concentrated acinar secretion product.
In conclusion, ACh-induced liquid secretion appears to be driven by
Cl and
secretion. Inhibition of
ACh-induced liquid secretion leads to the production of a relatively
dehydrated mucus, the rheological properties of which are altered in a
manner that predicts decreased mucus transportability and ciliary
clearance. These results are directly relevant to the pathogenesis of
CF in that they provide a physiological link between anion transport and alterations in the rheological properties of airway mucus.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-48622 and the Canadian Cystic Fibrosis Foundation.
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FOOTNOTES |
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Portions of this work have been published in abstract form (43).
Address for reprint requests: S. T. Ballard, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688.
Received 14 July 1997; accepted in final form 14 November 1997.
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