1 Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688; and 2 Schering-Plough Research Institute, Kenilworth, New Jersey 07033
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ABSTRACT |
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The present study was undertaken to identify and
determine the mechanism of noncholinergic pathways for the induction of
liquid secretion across airway epithelium. Excised porcine bronchi
secreted substantial and significant quantities of liquid when exposed to acetylcholine, substance P, or forskolin but not to isoproterenol, norepinephrine, or phenylephrine. Bumetanide, an inhibitor of Na+-K+-2Cl cotransport, reduced
the liquid secretion response to substance P by 69%. Approximately
two-thirds of bumetanide-insensitive liquid secretion was blocked by
dimethylamiloride (DMA), a Na+/H+ exchange
inhibitor. Substance P responses were preserved in airways after
surface epithelium removal, suggesting that secreted liquid originated
from submucosal glands. The anion channel blockers diphenylamine-2-carboxylate (DPC) and
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) inhibited >90% of
substance P-induced liquid secretion, whereas DIDS had no effect. DMA,
DPC, and NPPB had greater inhibitory effects on net
HCO
and HCO
cystic fibrosis transmembrane conductance regulator; bicarbonate; bumetanide; dimethylamiloride; cystic fibrosis
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INTRODUCTION |
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TRACHEOBRONCHIAL
SUBMUCOSAL GLANDS secrete numerous substances such as mucins,
which facilitate mucociliary clearance of inhaled debris, and various
antibacterial proteins including lysozyme, lactoferrin, and
-defensins that are critical to normal lung function (3, 20,
44). Normally, these proteinaceous substances are flushed from
gland ducts by cosecretion of liquid that is thought to originate from
serous cells that populate the distal ducts and demilunes of the
glands. Close coupling of liquid and macromolecule secretion ensures
that mucins are adequately hydrated for optimal ciliary transport by
surface epithelial cells and that antibacterial substances are
delivered to the airway surface in quantities sufficient to prevent
pathogen colonization.
Secretion of liquid from airway glands is principally under neural
control. Immunohistochemical and radioligand binding studies demonstrated that M3 muscarinic receptors (4),
1-adrenergic receptors (5),
2-adrenergic receptors (29), and substance P receptors (6) are present on airway submucosal gland
cells. Muscarinic or
-adrenergic receptor agonists substantially
enhance volume secretion from feline tracheal submucosal glands in vivo (36, 47). Intravenous administration of substance P also
greatly stimulates tracheal gland liquid secretion in domestic pigs,
suggesting that sensory afferents play a role in regulating submucosal
gland function (14). In cats,
-adrenergic receptor
stimulation produces a smaller liquid secretion response that varies
from mild to scant (26, 36).
Recent studies (2, 16, 45) provide significant insight
into the mechanisms responsible for liquid secretion in airway submucosal glands. Intact distal bronchi of pigs, when stimulated with
acetylcholine, secrete liquid through the active transport of both
Cl and HCO
and HCO
and
HCO
We considered the possibility that noncholinergic pathways might induce
gland liquid secretion by CFTR-independent mechanisms. The existence of
a CFTR-independent secretion pathway could be important therapeutically
in that it could be manipulated pharmacologically to bypass the
CFTR-dependent defect in gland liquid secretion that likely occurs in
CF. To identify these possible pathways, we screened numerous
secretagogues as potential stimulators of gland liquid secretion in
porcine bronchi. We found that liquid was secreted in response to both
muscarinic and substance P receptor agonists but that - and
-adrenergic receptor agonists produced no measurable effect. We
further report evidence that the liquid secretion response to substance
P closely mirrors that of acetylcholine in that it is driven by the
transepithelial secretion of both Cl
and
HCO
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METHODS |
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Experimental protocol. Young domestic pigs (~10-15 kg) were sedated with intramuscular xylazine (4 mg) and ketamine (80 mg) and euthanized with an intravenous overdose of pentobarbital sodium. The lungs were removed and placed in Krebs-Ringer bicarbonate (KRB) at room temperature. Distal bronchi were dissected from the surrounding tissue, and the side branches of the bronchi were ligated with sutures. Each bronchus was warmed slowly to 37°C (~0.1°C/min) in a KRB bath.
At the end of the warming period, the airways were removed from their bath solutions, and the lumens were cleared of all fluid and mucus. The air-filled bronchi were then cannulated with polyethylene tubing and returned to their respective baths. Either 100 µM acetylcholine (muscarinic receptor agonist), 1 µM substance P (neurokinin receptor agonist), 10 µM norepinephrine (agonist forCollection of airway liquid.
After a 2-h incubation with the secretion agonists, the bronchi were
removed from their cannulas and sectioned lengthwise, and all luminal
mucus liquid was recovered. Mucus liquid was placed into tared tubes
that were sealed and then weighed to determine secretion volume. Liquid
samples were frozen (70°C) for later analysis. Airway lengths and
outer diameters were measured and used to estimate luminal surface
areas as previously described (45). Net liquid secretion
rate (Jv) was calculated based on the total
secretion volume, the luminal surface area, and the time of agonist exposure.
Bicarbonate analysis.
Frozen samples were thawed to room temperature, and 1.4-10.0 µl
of each sample were added to 1,000 µl of Sigma INFINITY
CO2 reagent (Sigma). The solutions were mixed with a vortex
mixer for 15-20 s, and the absorbance of each sample was measured
at 380 nm with a Beckman DU-65 spectrophotometer. Blank samples
containing reagent and distilled H2O were assayed at the
beginning and end of the sample readings to allow correction for
temporal variance in the enzymatic reaction. Because the
HCO
Solution composition and drugs. KRB contained 112.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 2.4 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3, and 11.6 mM glucose. The pH of all KRB solutions was maintained at 7.4 by constant gassing with 95% O2-5% CO2. DPC (as N-phenylanthranilic acid) was purchased from Aldrich, and NPPB was obtained from Calbiochem. All other drugs were purchased from Sigma. Stock solutions of substance P and phosphoramidon were prepared in 0.9% saline. Stock solutions of norepinephrine, phenylephrine, and isoproterenol were prepared in deionized H2O, and stock solutions of all inhibitors and forskolin were prepared in DMSO. Equal volumes of the vehicle were added to all control tissues.
Statistics. The data are expressed as means ± SE. Statistical comparisons were made with paired t-tests, unpaired t-tests, or ANOVA, with either Dunnett's or Tukey's test for multiple comparisons when appropriate. Differences were considered significant when P < 0.05. The number of observations is indicated by n (animals).
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RESULTS |
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Untreated porcine bronchi secreted little liquid (1.9 ± 0.3 µl · cm2 · h
1;
n = 37; Fig. 1). However,
substantial liquid secretion occurred when the airways were exposed to
100 µM acetylcholine (16.9 ± 2.7 µl · cm
2 · h
1;
n = 6), 1 µM substance P (18.0 ± 1.0 µl · cm
2 · h
1;
n = 46), and 10 µM forskolin (8.5 ± 1.9 µl · cm
2 · h
1;
n = 6). No significant increase in
Jv was observed after treatment with either
-
or
-adrenergic agonists. Norepinephrine (10 µM), phenylephrine (10 µM), and isoproterenol (10 µM) induced secretions of only
2.5 ± 0.4 µl · cm
2 · h
1
(n = 4), 0.9 ± 0.4 µl · cm
2 · h
1
(n = 5), and 0.7 ± 0.5 µl · cm
2 · h
1
(n = 4), respectively. In a separate group of paired
tissues, the Jv response to substance P
(16.5 ± 2.8 µl · cm
2 · h
1;
n = 4) was unaffected by 10 µM atropine (19.4 ± 4.1 µl · cm
2 · h
1;
n = 4), indicating that the stimulatory effect was not
caused by secondary release of acetylcholine.
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When substance P-stimulated airways were pretreated with 100 µM DMA
to block HCO secretion,
substance P-induced liquid secretion was significantly reduced by 69%.
In the presence of both bumetanide and DMA, substance P-induced liquid
secretion fell by 89%, an inhibitory effect that was significantly
greater than that of bumetanide pretreatment alone. DPC (100 µM)
pretreatment, which was intended to block apical membrane anion
channels, nearly abolished the Jv response as
did pretreatment with 300 µM NPPB. Pretreatment with 1 mM DIDS, which
should have blocked Ca2+-activated anion channels,
outwardly rectifying anion channels, some volume-sensitive anion
channels, Cl
/HCO
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In a subset of airways, the [HCO secretion, increased
[HCO
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DPC pretreatment also nearly abolished
JHCO
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DISCUSSION |
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These studies show that substance P is an effective stimulator of
liquid secretion in porcine bronchi. Approximately 70% of the
Jv response to substance P was inhibited by
bumetanide, suggesting that active secretion of Cl
comprises the major driving force for liquid secretion. Most of the
bumetanide-insensitive Jv was blocked with DMA,
apparently by inhibiting active HCO
Previous studies demonstrate that substance P evokes liquid secretion from tracheal submucosal glands (14) and that afferent C fiber stimulation induces liquid secretion via an efferent vagal response (8). Together with a study (31) showing localization of substance P-containing neurons in the airways, these findings suggest the presence of 1) sensory afferent neurons that are capable of direct release of tachykinins at the site of stimulation within the airways and 2) efferent neurons passing through the vagus that release cholinergic neurotransmitters within airway tissues. The existence of this dual-excitation pathway ensures that a vigorous glandular secretion response will result from an appropriate mechanical or chemical stimulus of the airway mucosa. Copious secretion of both liquid and mucin from glands should aid mucociliary transport by increasing the volume of mucus liquid at the airway surface. Endogenous release of either acetylcholine or substance P in the vicinity of the surface epithelial cells should also increase ciliary beat frequency (49, 50), further accelerating mucociliary transport and facilitating removal of mucosal irritants.
A role for CFTR in secretion of airway gland liquid has been suspected
since the finding that serous cells of submucosal glands were a major
site of CFTR expression in human airways (11). Similar
distribution patterns occur in cows (19), ferrets
(39), and pigs (2). Reports (9,
40) that the Calu-3 cell, an airway serous cell line of human
origin, secretes Cl by a CFTR-dependent mechanism further
support this notion. Moon and colleagues (30) concluded
that CFTR is the exclusive Cl
conductive pathway
expressed in the apical membrane of Calu-3 cells, suggesting that anion
secretion in this cell type is critically dependent on the function of
this channel. In the present study, we conclude that CFTR mediates the
liquid secretion responses to substance P for the following reasons.
First, substance P-induced liquid secretion was abolished by both DPC
and NPPB, which are known to inhibit CFTR by blocking the ion pore from
the cytoplasmic side of the channel (51). However,
inhibition of secretion by DPC and NPPB does not alone confirm CFTR
involvement in this process because these agents are known to exert
nonselective effects that could complicate interpretation of their
actions. For instance, DPC has been shown to inhibit cyclooxygenase
(43), and NPPB has been shown to uncouple oxidative
phosphorylation in nonepithelial cells by acting as a protonophore
(28). Additionally, these agents block other anion
channels besides the CFTR (1, 18). The second reason to
suspect CFTR involvement is that the secretion response to substance P
is insensitive to DIDS, which does not inhibit the CFTR but does
inhibit both Ca2+-activated Cl
channels and
outwardly rectifying Cl
channels, the most likely
non-CFTR anion channels that would support transepithelial anion
secretion in postnatal airway epithelia (1). The third
piece of evidence implicating CFTR in gland secretion is the fluid
secretion response to acetylcholine and substance P that is partially
mimicked by forskolin, which increases intracellular cAMP by direct
stimulation of adenylyl cyclase. cAMP is a well-known activator of CFTR
(35). Finally, a role for CFTR in gland liquid secretion
is implied from studies of early CF disease (32, 52) where
mucoid obstruction of submucosal gland ducts is one of the earliest
signs of pathology in the lung. Similar mucin obstruction of gland
ducts can be reproduced in pig airways after inhibition of
Cl
and HCO
and
HCO
In the present study, virtually no liquid secretion resulted from
administration of isoproterenol, a -adrenergic receptor agonist.
Reports by others similarly suggest that
-agonists are weak
stimulators, at best, of gland liquid secretion. In studies with cat
tracheae, Leikauf and coworkers (26) reported that isoproterenol induced about half the secretion response of
acetylcholine, whereas Quinton (36) reported very little
effect of this agent. These findings are somewhat surprising, however,
in that
-agonists have been shown to stimulate CFTR-dependent
Cl
secretion in nasal epithelium, presumably through
elevation of intracellular cAMP (12). Our results do show
that liquid secretion is stimulated by forskolin, suggesting that the
secretion response is sensitive to cAMP. Therefore, it is likely that
-adrenoceptors are either sparse or absent on the gland cells that
are responsible for liquid secretion. Indeed, Mak and coworkers
(29) showed by radioligand binding that
2-adrenoceptors were present in airway submucosal gland
cells but that receptor density was much less than in airway surface
epithelium and alveolar walls.
We also saw no effect of -adrenergic agonists on airway liquid
secretion. This finding was unexpected because
-agonists have been
shown to evoke vigorous liquid secretion from feline tracheal glands in
vivo (36, 47). Similar to our studies, Joo and coworkers
(24) showed in preliminary studies that phenylephrine is a
poor agonist for liquid secretion by individual airway glands from
sheep tracheae. We speculate that these findings reflect species
differences in functional
-adrenergic receptor expression in airway
gland cells.
The mechanism of substance P-induced Cl and
HCO
90% of the liquid secretion responses to both agonists,
whereas DIDS has no effect. This pattern of responses is most
consistent with the mechanism for Cl
and
HCO
enters the
cell across the basolateral membrane by
Na+-K+-2Cl
cotransport and exits
across the apical membrane through the CFTR. Both
HCO
in the presence of bumetanide, a finding that is
inconsistent with conductance of both anions through a single channel.
Another possibility is that the composition of the secreted liquid is modified by the surface epithelium. For instance, acid equivalents could be added to the luminal liquid by H+ transporters in
the apical membrane during the 2-h incubation period. Indeed,
preliminary studies by Coakley and coworkers (7) suggest
that airway surface liquid is acidified with time through the actions
of H+-K+-ATPases expressed in the apical
membrane of the surface epithelium. Alternatively,
Cl
/HCO
. The actions of either transport
process could also account for the relatively low
[HCO
diffuses into the luminal liquid through the
paracellular junctions of the surface epithelium.
To sustain the electrical driving force for anion efflux across the
apical membrane, effective secretogogues must not only increase the
apical anion conductance but also increase the basolateral conductance
to K+ (42). When stimulated, both
M3 muscarinic receptors, which are the predominant
muscarinic receptor subtype in glands (4), and substance P
receptors have been shown in multiple systems to induce phospholipase
C-dependent inositol phospholipid hydrolysis and increase intracellular
Ca2+ concentrations (10, 13, 22, 23, 48). We
suspect that both of these agonists open Ca2+-activated
K+ channels in the basolateral membrane to augment the
electrical driving force for anion efflux across the apical membrane.
Sasaki and coworkers (37) demonstrated that acetylcholine
induces both Cl and K+ currents in airway
acinar gland cells by an inositol 1,4,5-trisphosphate-dependent process. Unfortunately, to date, we have been unable to identify, through the use of inhibitors, which specific subtype of K+
channel is involved in the liquid secretion response to either substance P or acetylcholine in porcine airways.
Our findings have particular relevance to the etiology of CF lung
disease. It is likely that liquid secretion from airway bronchial
glands induced by either substance P or acetylcholine, is dependent on
the CFTR. When the secretion of gland liquid is blocked, as occurs when
both Cl and HCO
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Angela Crews.
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FOOTNOTES |
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This work was funded by National Heart, Lung, and Blood Institute Grant HL-48622.
Address for reprint requests and other correspondence: L. Trout, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688 (E-mail: ltrout{at}bbl.usouthal.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 10 January 2001; accepted in final form 25 April 2001.
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