Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688
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ABSTRACT |
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In porcine
bronchi, inhibition of both
Cl and
HCO
3 transport is required to block
the anion secretion response to ACh and to cause mucus accumulation
within ACh-treated submucosal gland ducts [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]. In this previous study, a combination of
three potential HCO
3 transport
inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM
dimethylamiloride (DMA)] was used to block carbonic anhydrase,
Cl
/HCO
3
exchange, and
Na+/H+
exchange, respectively. The aim of the present study was to obtain a
better understanding of the mechanism of ACh-induced
HCO
3 secretion in airway glands by
determining which of the three inhibitors, in combination with
bumetanide, is required to block anion secretion and so cause ductal
mucin accumulation. Gland duct mucin content was measured in distal
bronchi isolated from domestic pigs. Addition of either bumetanide
alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no
significant effect on ACh-induced mean gland duct mucin content. In
contrast, glands treated with bumetanide plus DMA as well as glands
treated with all four anion transport blockers were almost completely
occluded with mucin after the addition of ACh. These data suggest that
mucin is cleared from the ducts of bronchial submucosal glands by
liquid generated from Cl
-
and DMA-sensitive HCO
3 transport.
acetylcholine; airway epithelium; bicarbonate secretion; bronchi; cystic fibrosis; mucus
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INTRODUCTION |
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THE VOLUME AND COMPOSITION of airway surface liquid is
thought to be regulated by active ion transport across airway
epithelia. Although Na+ absorption
and Cl secretion are the
two predominant transepithelial transport processes, a variety of
airway epithelia also secretes HCO
3 [nasal glands (6), trachea (18), bronchi (7), and Clara cells
(20)]. It is likely that HCO
3 is
secreted into the airway lumen through apical
Cl
channels such as the
cystic fibrosis (CF) transmembrane conductance regulator (CFTR) (18).
In the genetic disorder CF, CFTR is mutated and channel function is
disrupted (21). As a consequence, active secretion of
Cl
(2),
HCO
3 (18), and liquid (10) are probably impaired in CF airways.
A number of observations indicate that the submucosal glands may be
principally involved in the secretion of airway surface liquid. First,
submucosal glands secrete liquid at rest and in response to adrenergic
and cholinergic stimulation (8, 11, 15). Second, basal rates of active
Cl secretion are higher in
glandular airways (bronchi) than in aglandular airways (bronchioles)
(1). Third, pulmonary expression of the CFTR is highest in submucosal
glands (5). Fourth, one of the first signs of pulmonary pathology in CF
newborns is hypertrophy and mucus occlusion of the submucosal glands
(17).
Inglis et al. (7) recently showed that ACh, a stimulator
of gland mucus and liquid secretion, induces electrogenic anion secretion in excised porcine bronchi. After blockade of both
Cl and
HCO
3 secretion, ACh-induced anion
secretion is inhibited, and the submucosal gland ducts become occluded
with mucin (9). These studies suggest that both
Cl
and
HCO
3 secretion drive glandular liquid
secretion and that glandular liquid secretion is required to flush
secreted mucins out of the gland ducts. Because CFTR-mediated anion
secretion is defective in CF, it is likely that CF submucosal gland
ducts become occluded with mucus as a result of defective glandular anion and liquid secretion.
Because we initially considered that carbonic anhydrase,
Cl/HCO
3
exchange, and
Na+/H+
exchange could potentially be involved in airway epithelial
HCO
3 transport, the combination of
acetazolamide, DIDS, and dimethylamiloride (DMA) was used in previous
studies by Inglis et al. (7, 9) to block
HCO
3 secretion. The aim of the present study was to obtain a more precise understanding of the mechanism of
HCO
3 secretion from airway submucosal
glands by determining which of the three
HCO
3 secretion inhibitors is required,
in combination with bumetanide, to block ACh-induced anion secretion as
assessed by the mucin content of gland ducts in excised bronchi.
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METHODS |
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Airway excision. Young domestic pigs (8-18 kg) were obtained from a local vendor. The animals were sedated with intramuscular injections of ketamine (8 mg/kg) and xylazine (0.4 mg/kg) and killed with an intravenous overdose of pentobarbital sodium. Portions of the left and right lungs were rapidly excised and placed in Krebs-Ringer bicarbonate (KRB) solution at room temperature (25°C). Distal bronchi (external diameter 3-4 mm) were carefully dissected from the surrounding parenchyma, transferred to a tissue bath containing KRB solution, and slowly warmed (0.1-0.2°C/min) from room temperature to 37°C.
Histological analysis of glandular mucin
content. Isolated, warmed distal bronchi were cut into
1-cm lengths, and side branches were severed flush with the main airway
trunk. The bronchi were then incubated for 30 min in KRB solution
containing blockers of Cl
and HCO
3 secretion. Bumetanide (10 µM), a blocker of
Na+-K+-2Cl
cotransport, was used to inhibit
Cl
secretion. Blockers of
HCO
3 secretion were chosen based on
the model for airway epithelial HCO
3 secretion proposed by Smith and Welsh (18). According to this model,
HCO
3 and
H+ are produced from
CO2 and
H2O by intracellular carbonic
anhydrase. Protons are extruded from the cell interior by basolateral
Na+/H+
exchange, and HCO
3 exits across the
apical membrane through anion channels. The three
HCO
3 secretion blockers used in the
present study were acetazolamide (1 mM) to block carbonic anhydrase,
DMA (100 µM) to block
Na+/H+
exchange, and DIDS (1 mM) to block possible basolateral entry of
HCO
3 by
Cl
/HCO
3
exchange. Figure 1 shows a composite cell model of anion secretion and summarizes the intended targets for these
anion secretion inhibitors. To identify which
HCO
3 blockers were needed to inhibit
HCO
3 secretion, bronchi were treated
with either 1) bumetanide alone,
2) bumetanide and DMA,
3) bumetanide and acetazolamide,
4) bumetanide and DIDS, or
5) bumetanide and all three
HCO
3 secretion blockers. After
pretreatment with the anion transport blockers, 10 µM ACh was added,
and the tissues were incubated for 30 min. The bronchi were then
removed and placed immediately into Millonig's buffer containing 1%
paraformaldehyde and 1% glutaraldehyde at 4°C. Later, the tissues
were embedded in paraffin, cut into 5- to 6-µm-thick sections, and
stained for mucins with Alcian blue and pyronin Y at pH 2.5 as
previously described (9). This procedure stains sulfated glycoproteins
red-purple and sialylated glycoproteins blue but does not stain neutral
glycoproteins. We determined in parallel studies, however, that the
distribution of neutral glycoproteins in porcine bronchi closely
mirrors that of sulfated and sialylated glycoproteins (Inglis,
unpublished observations).
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A morphometric analysis was used to estimate the quantity of mucin within the submucosal gland ducts. All gland ducts within a single slide section were examined for each bronchus. The mucin content of each gland duct was scored in a blind fashion on a subjective linear scale from zero (no mucin in duct) to five (duct completely occluded with mucin). Each numerical increment between zero and five represents an ~20% increase in the ductal lumen area occupied by mucin.
Solution composition and drugs. The KRB solution contained (in mM) 112 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.6 glucose. Solution pH was maintained at 7.4 by frequent gassing with 5% CO2 in O2. All drugs and chemicals were purchased from Sigma. DMSO was used as the vehicle for bumetanide, acetazolamide, DIDS, and DMA.
Statistical analysis. Within an experimental group, each airway was obtained from a different animal. All data are reported as means ± SE. ANOVA with Tukey's post hoc test was used to compare mean values. P < 0.05 was considered the level of significance.
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RESULTS |
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Histological analysis of glandular mucin
content. To determine which anion transporters are
required to keep submucosal gland ducts clear of mucin, bronchi were
pretreated with various combinations of the anion transport blockers
before the addition of ACh. The gland duct mucin contents for each of
the five different treatments are shown in Figs.
2 and 3. Ductal mucin
content was scored on a linear scale from zero (duct contains no mucin)
to five (duct occluded with mucin; see
METHODS). Figure 2 shows the
frequency distribution of mucin scores, and Fig. 3 shows the average
mucin content of the gland ducts. In bronchi treated only with ACh, >50% of the ducts contained no measurable mucin (Fig. 2) and mean ductal mucin content was 0.66 ± 0.07 (Fig. 3). In contrast, gland ducts from bronchi that were pretreated with all four anion transport blockers before ACh treatment contained significantly greater quantities of mucin (3.60 ± 0.09; Fig. 3). Forty percent of the ducts in these bronchi were completely occluded with mucin (Fig. 2).
Mucin content of gland ducts pretreated with either bumetanide alone
(1.33 ± 0.46), bumetanide and acetazolamide (1.95 ± 0.45), or
bumetanide and DIDS (1.32 ± 0.45) was not significantly different from gland ducts treated only with ACh (Fig. 3). In contrast, the mucin
content of gland ducts that were pretreated with bumetanide and DMA
(3.61 ± 0.21) was comparable to the mucin content of gland ducts
pretreated with all four anion transport blockers (Fig. 3) and was
significantly greater than tissues that were pretreated with either no
inhibitor, bumetanide alone, bumetanide and acetazolamide, or
bumetanide and DIDS. Forty-one percent of the ducts that were pretreated with bumetanide and DMA were completely occluded with mucin
(Fig. 2). These results confirm previous findings that glands pretreated with Cl and
HCO
3 secretion blockers become
occluded with mucin when ACh is added (9). Photomicrographs of tissues pretreated with either bumetanide and DMA or the DMSO vehicle before
the addition of ACh illustrate the extent of gland duct occlusion
induced by these agents (Fig. 4).
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DISCUSSION |
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A previous study (9) of distal bronchi provided indirect evidence that
1)
Cl and
HCO
3 secretion drives the glandular
liquid secretion response to ACh and
2) ACh-induced glandular liquid secretion can be uncoupled from mucus secretion by pretreatment with
blockers of both Cl
(bumetanide) and HCO
3 (acetazolamide,
DMA, and DIDS) transport. Unfortunately, this previous study provided little insight into the mechanism of
HCO
3 secretion. In the present study,
measurements of gland duct mucin content were used to determine which
of the three HCO
3 secretion blockers
is specifically required to inhibit ACh-induced, HCO
3- dependent liquid secretion.
Pretreatment with either acetazolamide or DIDS, in combination with
bumetanide, did not result in mucin accumulation after the addition of
ACh. In contrast, DMA plus bumetanide was as effective as the cocktail of all three HCO
3 blockers plus
bumetanide at causing gland duct mucin accumulation after addition of
ACh. These results suggest that DMA-sensitive
Na+/H+
exchange is critical for glandular
HCO
3 and liquid secretion.
The finding that glands pretreated with DMA become occluded with mucin
after addition of ACh is consistent with a major role for
Na+/H+
exchange in airway gland HCO3
secretion. Similar models of HCO
3
secretion that require
Na+/H+
exchange have been described in a variety of tissues including pancreatic ducts, ileum, and salivary acini (3, 13, 14). Muscarinic
agonists are known to stimulate
Na+/H+
exchange in acinar cells of the pancreas and salivary, lacrimal, and
nasal glands (4, 6, 16, 22). The mechanism for ACh-induced stimulation
of the
Na+/H+
exchanger has not been fully determined, but elevation of intracellular calcium concentration is likely to play a role (22). An alternative explanation for the DMA-induced mucin occlusion of submucosal gland
ducts could be that DMA stimulates gland mucin secretion. However, in a
previous study, Inglis et al. (9) demonstrated that the combination of
DMA, acetazolamide, and DIDS had no effect on gland duct mucin content
in the absence of bumetanide. If DMA indeed stimulated mucin secretion,
we would have expected an increase in ductal mucin content under those
conditions. This previous study also demonstrated that the effect of
the HCO
3 blockers on gland mucin
secretion is reversible, indicating that the blockers are unlikely to
be toxic to the tissues.
The inability of DIDS to affect gland duct mucin content suggests that
Cl/HCO
3
exchange activity is low or absent from airway glandular epithelium.
Taken together, the effects of DMA and DIDS on ductal mucin
accumulation suggest that the majority of secreted
HCO
3 is generated within the cell cytoplasm and that little or no HCO
3
enters the cells across the basolateral membrane (Fig. 1). It is
surprising, therefore, that acetazolamide did not induce glandular
mucin accumulation. It is possible that carbonic anhydrase was
incompletely blocked despite the presence of acetazolamide or that the
uncatalyzed generation of intracellular
HCO
3 supported sufficient liquid
secretion to flush mucin from the gland ducts. Ductal mucin content is
unlikely to be a truly quantitative measure of gland liquid secretion.
A threshold level of liquid secretion is probably required to flush
mucin from the gland ducts. If gland liquid secretion is only partially
inhibited, mucin may still be adequately cleared.
Evidence for HCO3 secretion has been
found in nasal glands (6) as well as in tracheal (18), bronchial (7),
and bronchiolar epithelia (20). Although its physiological role has not
yet been determined, airway epithelial
HCO
3 secretion may play an important
role in the regulation of airway surface liquid pH. Because both the
physical properties of mucus (19) and the activity of cilia (12) are
sensitive to changes in H+
concentration, mucociliary clearance could be strongly influenced by
the regulation of airway surface liquid pH.
In summary, this study suggests that ACh-induced
HCO3 secretion in airway glandular
epithelium is critically dependent on basolateral
Na+/H+
exchange. These findings reinforce the previous suggestion by Inglis et
al. (9) that ACh-induced liquid secretion from airway submucosal glands
is driven by both Cl
and
HCO
3 secretion.
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ACKNOWLEDGEMENTS |
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We thank Dr. Walter Wilborn and Barbara Hyde of the Structural Research Center (Mobile, AL) for performing the histology.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-48622.
Address for reprint requests: S. T. Ballard, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688.
Received 23 April 1997; accepted in final form 2 February 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ballard, S. T.,
J. D. Fountain,
S. K. Inglis,
M. R. Corboz,
and
A. E. Taylor.
Chloride secretion across distal airway epithelium: relationship to submucosal gland distribution.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L526-L531,
1995
2.
Boucher, R. C.,
M. J. Stutts,
M. R. Knowles,
L. Cantlety,
and
J. T. Gatzy.
Na+ transport in cystic fibrosis respiratory epithelia.
J. Clin. Invest.
78:
1245-1252,
1986[Medline].
3.
Case, R. M.,
and
B. E. Argent.
Pancreatic duct cell secretion. Control and mechanisms of transport.
In: The Pancreas: Biology, Pathobiology and Disease (2nd ed.), edited by V. L. W. Go,
E. P. DiMagno,
J. D. Gardner,
E. Lebenthal,
H. A. Reber,
and G. A. Scheele. New York: Raven, 1993, p. 301-350.
4.
Dissing, S.,
and
B. Nauntofte.
Na+ transport properties of isolated rat parotid acini.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G1044-G1055,
1990
5.
Engelhardt, J. F.,
J. R. Yankaskas,
S. A. Ernst,
Y. Yang,
C. R. Maino,
R. C. Boucher,
J. A. Cohn,
and
J. M. Wilson.
Submucosal glands are the predominant site of CFTR expression in the human bronchus.
Nat. Genet.
2:
240-248,
1992[Medline].
6.
Ikeda, K.,
M. Ishigaki,
D. Wu,
H. Sunose,
and
T. Takasaka.
Na+ transport processes in isolated guinea pig nasal gland acinar cells.
J. Cell. Physiol.
163:
204-209,
1995[Medline].
7.
Inglis, S. K.,
M. R. Corboz,
A. E. Taylor,
and
S. T. Ballard.
Regulation of ion transport across porcine distal bronchi.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L289-L297,
1996
8.
Inglis, S. K.,
M. R. Corboz,
A. E. Taylor,
and
S. T. Ballard.
In situ visualization of bronchial submucosal glands and their secretory response to acetylcholine.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L203-L210,
1997
9.
Inglis, S. K.,
M. R. Corboz,
A. E. Taylor,
and
S. T. Ballard.
Effect of anion transport inhibition on mucus secretion by airway submucosal glands.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L372-L377,
1997
10.
Jiang, C.,
W. E. Finkbeiner,
J. H. Widdicombe,
P. B. McCray, Jr.,
and
S. S. Miller.
Altered fluid transport across airway epithelium in cystic fibrosis.
Science
262:
424-427,
1993[Medline].
11.
Leikauf, G. D.,
I. F. Ueki,
and
J. A. Nadel.
Autonomic regulation of viscoelasticity of cat tracheal gland secretions.
J. Appl. Physiol.
56:
426-430,
1984
12.
Luk, C. K.,
and
M. J. Dulfano.
Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants.
Clin. Sci.
64:
449-451,
1983[Medline].
13.
Minhas, B. S.,
S. K. Sullivan,
and
M. Field.
Bicarbonate secretion in rabbit ileum: electrogenicity, ion dependence and effects of cyclic nucleotides.
Gastroenterology
105:
1617-1629,
1993[Medline].
14.
Nauntofte, B.
Regulation of electrolyte and fluid secretion in salivary acinar cells.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G823-G837,
1992
15.
Quinton, P. M.
Composition and control of secretions from tracheal bronchial submucosal glands.
Nature
279:
551-552,
1979[Medline].
16.
Saito, Y.,
T. Ozawa,
and
A. Nishiyama.
Acetylcholine-induced Na+ influx in the mouse lacrimal gland acinar cells: demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements.
J. Membr. Biol.
98:
135-144,
1987[Medline].
17.
Sheppard, M. N.
The pathology of cystic fibrosis.
In: Cystic Fibrosis, edited by M. E. Hodson,
and D. M. Geddes. London: Chapman & Hall, 1995, p. 131-149.
18.
Smith, J. J.,
and
M. J. Welsh.
cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia.
J. Clin. Invest.
89:
1148-1153,
1992[Medline].
19.
Tam, P. Y.,
and
P. Verdugo.
Control of mucus hydration as a Donnan equilibrium process.
Nature
292:
340-342,
1981[Medline].
20.
Van Scott, M. R.,
C. M. Penland,
C. A. Welch,
and
E. Lazarowski.
-Adrenergic regulation of Cl- and HCO
3 secretion by Clara cells.
Am. J. Respir. Cell Mol. Biol.
13:
344-351,
1995[Abstract].
21.
Welsh, M. J.,
and
A. E. Smith.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[Medline].
22.
Zhao, H.,
and
S. Muallem.
Agonist-specific regulation of [Na+]i in pancreatic acinar cells.
J. Gen. Physiol.
106:
1243-1263,
1995[Abstract].