Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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We previously reported that substance P (SP) and
ATP evoke transient, epithelium-dependent relaxation of mouse tracheal
smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl secretion across epithelial monolayers, we tested the
hypothesis that epithelium-dependent relaxation of mouse trachea
depends on Cl
channel function. In perfused mouse
tracheas, the responses to SP and ATP were both inhibited by the
Cl
channel inhibitors diphenylamine-2-carboxylate and
5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was
unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and
relaxation to SP was unaffected by either DIDS or DNDS. Replacing
Cl
in the buffer solutions with the impermeable anion
gluconate on both sides of the trachea inhibited relaxation to SP or
ATP. In contrast, increasing the gradient for Cl
secretion using Cl
-free medium only in the tracheal lumen
enhanced the relaxation to SP or ATP. We conclude that Cl
channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS
suggested the involvement of a DIDS-insensitive Cl
channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl
channel. To test this hypothesis, we
evaluated tracheas from CFTR-deficient mice and found that the peak
relaxation to SP or ATP was not significantly different from those
responses in wild-type littermates. This suggests that a
DIDS-insensitive Cl
channel other than CFTR is active in
the SP response. This work introduces a possible role for
Cl
pathways in the modulation of airway smooth muscle
function and may have implications for fundamental studies of airway
function as well as therapeutic approaches to pulmonary disease.
smooth muscle; substance P; adenosine triphosphate; trachea; mouse
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INTRODUCTION |
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THE EPITHELIAL CELLS
LINING the airways are the first surface exposed to inhaled
compounds and may play a major role in determining the airway response
to various stimuli. The epithelium may function as a source of relaxing
factors or as a simple barrier against diffusion of inhaled agents
(21). We have recently reported that substance P (SP) and
ATP evoke transient, epithelium-dependent relaxations of constricted
mouse trachea (10). These are likely to be physiologically
relevant responses, since SP- immunoreactive fibers are present in
mouse airways (28) and ATP is normally secreted into the
airway lumen by the epithelium (15). Since both SP and ATP
are also known to elicit transepithelial Cl secretion in
cultured epithelial monolayers (2, 24), it is
possible that these two responses are functionally related. We thus
tested the hypothesis that epithelium-dependent relaxation of mouse
trachea depends on Cl
channel function. We used the mouse
because it offers a powerful system in which the function of specific
proteins may be investigated using gene-targeting strategies or
transgenic approaches (22).
To study epithelial function in intact airways, we used an apparatus
for evaluating constriction and relaxation responses in isolated,
perfused mouse tracheas (10). This allowed side-specific delivery of inhibitors to either the airway lumen or smooth muscle. The effects of four Cl channel inhibitors,
DIDS, 4, 4'-dinitrostilbene-2,2'-disulfonic acid (DNDS),
diphenylamine-2-carboxylate (DPC), and
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), and
Cl
substitution were studied in tracheas from wild-type
mice. Based on our results in this part of the study and on the known
sensitivity of cystic fibrosis transmembrane conductance regulator
(CFTR) to these inhibitors (1, 11, 16), we further
hypothesized that the observed relaxation to SP may be mediated through
the CFTR Cl
channel. To test this second hypothesis, we
used a genetic approach and studied tracheal function in a mouse
line in which the CFTR channel has been deleted by gene targeting
through homologous recombination [CFTR(
/
), purchased as
cftrtm1Unc from The Jackson Laboratory,
Bar Harbor, ME].
While Cl currents appear to be involved in the response
to osmotic stimuli (6), the present study offers the first
evidence for the involvement of Cl
channels in
receptor-mediated, epithelium-dependent modulation of airway smooth
muscle contractility. This may have implications for fundamental
studies of airway function as well as for therapeutic approaches to
pulmonary disease.
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MATERIALS AND METHODS |
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Tracheal tissue preparation.
Mice aged 10-14 wk were selected for experimentation. Mice were
killed by CO2 asphyxiation, and the trachea was removed and prepared as described below. Excised tracheas were maintained in
physiological salt solution (PSS) of the following composition (in mM):
118 NaCl, 4.73 KCl, 1.2 MgSO4, 0.026 EDTA, 1.2 NaH2PO4, 2.5 CaCl2, and 11 glucose,
buffered with 25 NaHCO3 to attain a pH of 7.4 at 37°C
when bubbled with a mixture of 95% O2-5% CO2. For Cl substitution studies, all
Cl
-containing salts were replaced with the same
concentration of gluconate salts. When changing to
Cl
-free PSS, solutions were changed at least four times
during the course of 1 h before the trachea was stimulated.
Perfused trachea preparations.
The technique for perfusing an isolated mouse trachea with separate
solutions bathing the smooth muscle and epithelium has been previously
described in detail (10). Briefly, each trachea was tied
with the ends around two cannulas. Differential pressure (P) across
the trachea was measured from wall taps near the cannula openings using
pressure transducers (Cobe, Lakewood, CO) and recorded using the BioPac
data acquisition system (BioPac Systems, Goleta, CA). At a perfusion
rate of 16 ml/min, baseline
P was 1.10 ± 0.09 mmHg
(n = 15) for tracheas without stimulation.
Force measurements in tracheal ring preparations.
Tracheal rings were mounted isometrically to force transducers as
previously described in detail (10). Paired tracheas from CFTR(/
) and wild-type mice were mounted in the same chamber. Both
epithelial and smooth muscle surfaces were bathed by the same buffer
solution and thus exposed to identical concentrations of inhibitors or
test agents.
Pharmaceuticals. Unless otherwise noted, chemicals were obtained from Sigma (St. Louis, MO). Acetylcholine chloride (ACh), SP, ATP, and DNDS (Molecular Probes, Eugene, OR) were dissolved in distilled water to produce concentrated stocks. DPC (Fluka, Milwaukee, WI), PGE2 (Cayman Chemical, Ann Arbor, MI), and indomethacin were dissolved in ethanol. DIDS and NPPB were dissolved in DMSO (Calbiochem, San Diego, CA). When added to the buffer solution, the final concentration of ethanol or DMSO was <1% vol/vol. In control experiments, this concentration of ethanol or DMSO alone had no effect on airway constriction or relaxation.
Data analysis. Data are expressed as means ± SE. The n value indicates the number of mice used. Standard ANOVA was used, with P < 0.05 taken as evidence of statistical significance. Concentration-response curves were fit with a logistic equation using Origin data analysis software (Microcal Software, Northampton, MA).
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RESULTS |
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Epithelium-dependent relaxation in perfused mouse trachea.
In each experiment, control responses to luminal SP and ATP were
measured in perfused tracheas constricted with 10 µM ACh in the
extraluminal bathing solution (Fig.
1A). This
concentration of ACh is approximately the ED80 for mouse
trachea (10) and resulted in a repeatable constriction
that increased P by 1.19 ± 0.13 mmHg (n = 15)
above baseline and was stable within 5 min of ACh addition. After
tracheas were constricted, 10 nM SP was added to the luminal perfusate,
and a transient relaxation was observed. The full magnitude of
contraction was recovered within 15 min, at which time 10 µM ATP was
added to the luminal perfusate and a second transient relaxation was
observed. This was done without changing the luminal perfusate or the
extraluminal bathing solution to maintain the same conditions and
facilitate comparison of the SP and ATP responses.
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Time control studies.
Because our experimental design incorporated multiple measurements, we
determined the reproducibility of mouse tracheal responses in tracheal
rings from six mice. Ten cycles of contraction to ACh followed by
treatment with SP or ATP and then subsequent treatment with the other
relaxant (ATP or SP) were performed (Fig.
2). For the first five cycles and the
final cycle, SP was given before ATP. For cycles 6-9, ATP
was given before SP. Contraction to 10 µM ACh showed a decreasing
trend over 10 h; however, the force was only significantly less
for the contraction 8 h after dissection. The subsequent
contraction was not different from any of the previous contractions.
Relaxation to 10 nM SP depended on the time between treatments. This is
not unexpected, since relaxation to SP is known to be tachyphylactic
(10). With only 5 min between washout of the previous SP
treatment and the subsequent contraction cycle, relaxation to SP was
significantly reduced. Longer periods between SP exposures
increased the relaxation to SP, such as occurred when ATP was given as
the first relaxant and in the final cycle. While some variability in
the SP and ATP responses was observed, there was no significant loss of
relaxation to SP or ATP over the 10-h time control, with the exception
of the second SP treatment following the shorter (5-min) washout
period.
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Effects of Cl channel inhibitors on
epithelium-dependent relaxation.
After the control measurements and subsequent 10-min washout period,
inhibitors were added to the luminal perfusate or extraluminal bath as
specified. DIDS, DNDS, and DPC were added 15 min and NPPB 20 min before
the trachea was constricted and remained present for the duration of
the experiment. The maximum concentrations of DIDS (1 mM) and DPC (2.5 mM) were chosen based on reported concentrations that effectively
inhibit Cl
channels (5, 14, 17, 26, 30).
NPPB (10 µM) blocked relaxation of tracheal rings to SP and ATP
(n = 7). Ten micromolar NPPB was less effective at
blocking the relaxation responses in perfused tracheas; thus 30 µM
was used (Fig. 1B). The relaxation to ATP was blocked by
DIDS, DPC, and NPPB but not by DNDS (tracheal rings constricted with 10 µM ACh relaxed 69 ± 4 vs. 54 ± 3% in controls,
n = 6). NPPB inhibited relaxation to ATP more
effectively when delivered to the luminal perfusate than when
administered to the outer bathing solution only (Fig.
3A). Inhibition by DPC or NPPB was reversible on replacement of the PSS. DIDS inhibition of
ATP relaxation was only partially reversible, even following a 1-h
recovery period. In contrast to the ATP response, the relaxation to SP
was unaffected by DIDS or DNDS (62 ± 3 vs. 71 ± 4% in controls, n = 6) but was blocked by DPC and NPPB. As with the ATP
response, 30 µM NPPB in the lumen blocked the relaxation to SP more
effectively than extraluminal NPPB (Fig. 3B).
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Effects of Cl substitution in perfused
tracheas.
An additional method for inhibiting Cl
currents is to
replace Cl
with an impermeable anion such as gluconate
(11). To obtain completely Cl
-free
conditions, potassium gluconate rather than ACh was used to depolarize
and constrict the smooth muscle. In Cl
-free PSS, the
constriction to 40 mM potassium gluconate was <50% of control on
average. In control experiments (n = 3), we
demonstrated that both SP and ATP could relax tracheas constricted with
40 mM potassium gluconate (Fig.
4A). Figure 4B
shows the typical inhibitory effects of Cl
-free PSS on
the relaxation to SP or ATP. Cl
-free PSS significantly
inhibited the relaxation to SP (24 ± 12% of constriction vs.
61 ± 8% in controls) or ATP (20 ± 7% of constriction vs.
60 ± 9% in controls). Consistent with the results obtained with
Cl
channel inhibitors, the studies in
Cl
-free PSS suggest a linkage between Cl
currents and epithelium-dependent relaxation.
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Epithelium-dependent relaxation with
Cl-free PSS in the lumen
only.
If Cl
channel currents are functionally linked to the
epithelium-dependent relaxation to SP or ATP, one possible way to
enhance the responses would be to increase the gradient for
Cl
secretion using Cl
-free PSS in the
airway lumen only. We also used submaximal concentrations of SP or ATP
to avoid potential saturation of the responses. Constriction to 10 µM
ACh was not affected when Cl
-free PSS was perfused
through the lumen (
P = 0.90 ± 0.10 mmHg with luminal
Cl
-free PSS vs. 0.95 ± 0.10 mmHg with regular PSS;
n = 8). As shown in Fig.
5, Cl
-free PSS only in the
lumen enhanced relaxation to 1 nM intraluminal SP or 0.3 µM
intraluminal ATP. Relaxation to SP was increased by 31% above control
(relaxation with luminal Cl
-free PSS was 60 ± 8 vs.
46 ± 10% in control, P < 0.05;
n = 8), and relaxation to ATP was increased by 43% (73 ± 3 vs. 51 ± 8% in control, P < 0.02;
n = 7).
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PGE2 relaxation.
The inhibition of these epithelium-dependent relaxations by
interventions designed to affect Cl currents could also
be attributed to direct effects on the ability of the smooth muscle to
relax. To provide a positive control for relaxation when responses to
both SP and ATP were blocked, constricted tracheas were treated with
PGE2. Smooth muscle-specific relaxation was elicited by
addition of PGE2 only to the extraluminal bath. The
concentrations of PGE2 were chosen because they produced a relaxation similar to that of SP or ATP. Perfusing the lumen with DPC
had a small but statistically significant effect on relaxation to 30 nM
PGE2 and no effect on relaxation to 100 nM
PGE2. Ten micromolar NPPB slightly decreased relaxation to
PGE2 in tracheal rings (Fig.
6), and this effect was not reversible on
washout of NPPB, in contrast to relaxation to SP or ATP. While the
Cl
channel inhibitors nearly abolished the
epithelium-dependent responses to SP or ATP (reducing them to
2-26% of control), the smooth muscle relaxation to
PGE2 was only moderately affected (73-93% of
control). Cl
-free PSS did not affect the sensitivity of
the concentration response to PGE2 (IC50 = 104 ± 67 nM compared with 15 ± 1 nM in control) but marginally
reduced the maximum relaxation (84% of relaxation was retained
compared with control). Thus the effects of Cl
-free PSS
and the various Cl
channel inhibitors do not exert their
major effects at the smooth muscle level.
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Isometric force measurements in tracheal rings from
CFTR-deficient mice.
The CFTR Cl channel is blocked by DPC but is unaffected
by DIDS (1). Our observation that the relaxation to SP was
also blocked by DPC but not by DIDS suggested the potential involvement of the CFTR Cl
channel. Tracheas from CFTR(
/
) mice
were paired with wild type and were mounted in the same bath for
isometric force measurements. There were no differences between
CFTR(
/
) and wild-type tracheas in gross morphological properties
(Table 1) or constriction to ACh (Table
2).
After constriction with 10 µM ACh, tracheal pairs were treated with
SP or ATP. In wild-type trachea, DIDS inhibited the relaxation to ATP
but not that to SP (Fig. 7), in
agreement with the studies on the perfused trachea. NPPB (10 µM)
blocked the relaxation both to SP and to ATP in a reversible manner.
The relaxations to SP and ATP persisted in the tracheas from
CFTR(
/
) mice, and the magnitude of relaxation was not significantly
different from that in the wild type (Fig. 7). It was not possible to
study the effects of DPC in the tracheal ring preparation because 2.5 mM DPC completely blocked contraction to ACh (n = 4).
As with DPC blockade of the SP or ATP relaxations, the blockade of
contraction was reversible. Normal contractions to ACh were observed 30 min after DPC washout.
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DISCUSSION |
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The role of the airway epithelium in modifying the contractility
of the underlying smooth muscle is not fully understood. It has been
suggested that the primary function of the epithelium is to provide a
barrier of protection between the airway smooth muscle and inhaled
irritants (21). More recently, studies have demonstrated
that the epithelium can be an active source of mediators that relax
constricted airways (7, 10, 27). SP and ATP are two
agonists that stimulate an epithelium-dependent relaxation of
constricted airways (10). Both of these agonists have also been shown to stimulate Cl secretion across the airway
epithelium into the airway lumen. The first objective of this study was
to investigate whether these two actions were functionally linked.
Specifically, is Cl
channel activity necessary for SP or
ATP to elicit epithelium-dependent relaxation of airways?
To test the hypothesis that the epithelium-dependent relaxation to SP
and ATP depends on Cl channel activity, we evaluated the
effects of various pharmacological inhibitors of Cl
channels on SP and ATP responses. The concentrations of
Cl
channel inhibitors were chosen based on published
values shown to effectively block Cl
channels (5,
11, 14, 17, 26, 30). Although the specificity of
Cl
channel inhibitors is not absolute, the combined
evidence from our studies with several different inhibitors or
Cl
substitution supports a dependence on Cl
currents for receptor-mediated, epithelium-dependent relaxation.
Of the inhibitors tested, NPPB is the most potent Cl
channel inhibitor (29) and is believed to be the most
selective for Cl
channels. To investigate whether
epithelial or smooth muscle Cl
currents are more
important in the relaxation to SP or ATP, we used side-specific
delivery of NPPB to either the airway lumen or outer perfusate in our
perfused trachea system. We first used 10 µM NPPB, since this
concentration effectively blocked relaxation of tracheal rings to SP
and ATP. Interestingly, when delivered intraluminally, extraluminally,
or bilaterally, 10 µM NPPB was less effective at blocking the
relaxation to SP or ATP in the perfused trachea than it was in tracheal
rings. Bilateral treatment with NPPB should be equivalent to the
conditions in the isometric force bath, with the exception of constant
flow through the airway lumen. Thus it appears that the effectiveness
of NPPB blockade may depend on flow across the epithelium. At 30 µM,
intraluminal or extraluminal NPPB inhibited relaxation to SP or ATP;
however, the inhibitor was greater than twofold more effective (Fig. 2) when delivered luminally. This finding suggests that blockade of
epithelial Cl
channels is more important than blockade of
smooth muscle Cl
channels for inhibiting
epithelium-dependent relaxation to SP or ATP.
We also used nominally Cl-free solutions in which
Cl
has been replaced with an anion that does not pass
easily through Cl
channels to test whether the SP and ATP
responses depend on Cl
currents. Such techniques have
been successfully used in experiments on cultured epithelial cells;
however, the use of intact trachea in our experimental system makes
interpretation of these data more complex. To completely remove
Cl
in the PSS that superfuses the smooth muscle in some
experiments, we changed the constricting stimulus to potassium
gluconate, since ACh was typically used to constrict the trachea in
PSS. Smooth muscle contraction was reduced in Cl
-free
PSS. Cl
is an important intracellular anion in smooth
muscle, and removing Cl
from the system may directly
impair smooth muscle contraction and/or relaxation.
The reduced contractions in the absence of Cl might be
due to stimulation of the release of a relaxing prostanoid.
Contractions to ACh were reduced in Cl
-free PSS, a
finding similar to that reported for rat tracheal smooth muscle
(25). Indomethacin did not prevent the reduction in airway
contractility in Cl
-free PSS, thereby excluding
eicosanoid release as a mechanism to explain the effects of
Cl
-free PSS on airway constriction. Because
Cl
-free PSS reduced contraction (Table 2), direct
comparison of its blockade of SP and ATP to their effects in control
PSS is not possible. However, since lower levels of constriction are generally associated with greater effectiveness of relaxing agents, the
finding that Cl
substitution inhibits rather than
augments the relaxation to SP or ATP further supports the conclusion
that epithelium-dependent relaxation depends on Cl
channel activity.
The effect of DIDS on epithelium-dependent relaxation to ATP may be due
to DIDS blockade of purinoceptors (4). DNDS is similar to
DIDS in its effects on Cl channels (3) yet
does not block purinoceptors (20). We found that DNDS did
not block relaxation to either SP or ATP. Regardless of the effects of
DIDS on ATP-induced relaxation, the important finding from the DIDS
experiments is that the response to SP is not sensitive to DIDS inhibition.
It is possible that the interventions used to inhibit Cl
channels blocked relaxation at the smooth muscle level. For the studies using DIDS, relaxation to SP provided a valuable internal positive control for relaxation, since SP relaxation was unaffected in the
presence of DIDS. In the cases where relaxation to both SP and ATP was
blocked, we investigated the effects of these inhibitors on relaxation
to exogenous PGE2. PGE2 was chosen as a
positive control since relaxation to SP or ATP is likely to involve an eicosanoid (10), and PGE2 is a possible
mediator of SP relaxation (23). In tracheas where
relaxation to both SP and ATP were blocked by Cl
channel
inhibitors or Cl
-free PSS, 100 nM PGE2
relaxed constricted tracheas similar to the control responses to SP or
ATP. One interesting finding was that NPPB reduced the response to
PGE2, although not to the extent that relaxation to SP or
ATP was blocked. This effect on relaxation to PGE2 did not
reverse on removal of NPPB; however, relaxation to SP or ATP recovered
in these same tracheas after NPPB washout. This suggests that the
irreversible inhibition of NPPB on relaxation to PGE2 was
different from its effects on relaxation to SP or ATP. For relaxation
to PGE2 in Cl
-free PSS, the shift in
sensitivity may be due to alterations in smooth muscle pH. Surrounding
smooth muscle with gluconate-substituted PSS has been reported to raise
intracellular pH (12), and alkalinization of smooth muscle
shifts the sensitivity to certain relaxation stimuli (18).
One Cl channel that has been characterized based on its
sensitivity to DPC while being insensitive to DIDS is the CFTR
Cl
channel (1). Our results suggested that
the epithelium-dependent relaxation to SP may be mediated through the
CFTR channel. We tested this hypothesis using mice deficient in CFTR
(19). These mice develop an intestinal pathology similar
to that of the human disease cystic fibrosis, but there is no overt
phenotype reported in the lungs of CFTR(
/
) mice (8).
Measurements of isometric force were used to compare tracheal rings
from CFTR(
/
) and wild-type mice in the same PSS and exposed
to identical concentrations of test agents. There were no differences
in the ACh concentration-force relationship for CFTR(
/
) and
wild-type tracheas. The finding that SP-induced relaxation was intact
in the CFTR(
/
) trachea indicated that CFTR function is not
required for airway relaxation to SP. The SP relaxation may be mediated
through other Cl
channels based on the documented
persistence of DIDS-insensitive Cl
currents in the
trachea from CFTR(
/
) mice (9).
The exact mechanism by which epithelium-dependent relaxation involves
Cl channels is not known. We have previously reported
that indomethacin irreversibly blocks the relaxation to SP and ATP
(10). It is presumed that this effect is due to blockade
of cyclooxygenase-dependent production of an eicosanoid relaxing
factor. Recent studies have also suggested a possible role for
Cl
currents in epithelium-dependent relaxation to osmotic
stimuli (6); however, the airway response to osmotic
stimuli is not inhibited by indomethacin, unlike our observed responses
to SP and ATP (10). We found no reports of indomethacin,
DPC, or NPPB interfering directly with the receptors for SP or ATP.
Although the specific mechanism by which Cl movement
facilitates epithelium-dependent airway relaxation remains to be
elucidated, the current study offers the first evidence for a role of
Cl
currents in epithelium-mediated relaxation of mouse
airways. Since both the epithelial and smooth muscle components may be affected by the experiments demonstrating the dependence on
Cl
currents, one cannot unambiguously assign a specific
cellular locus for the effect. However, some of the observed results
suggest that epithelial Cl
channels are of greater
importance in the epithelium-dependent relaxation to SP or ATP. First,
the relatively impermeable Cl
channel inhibitor NPPB was
more effective when delivered luminally compared with when superfused
on the smooth muscle. Additionally, the finding that DPC blocks
constriction of tracheal rings combined with the fact that DPC in the
lumen only did not affect constriction in perfused trachea suggests
that little or no DPC reached the smooth muscle, and thus the major
site of its effects was most likely the epithelium. Finally, the
strongest evidence to support a dependence on epithelial
Cl
channels was the finding that Cl
-free
PSS in only the lumen enhanced the relaxation to SP or ATP. Under these
conditions, the smooth muscle was not exposed to Cl
-free
conditions; however, the Cl
gradient for epithelial
Cl
secretion would be increased. This did not appear to
alter any basal release of mediators from the epithelium, since
constriction to ACh was not affected by luminal Cl
-free
PSS. Based on the combined evidence from these three experiments, it is
likely that our results reflect a greater influence of epithelial Cl
currents.
This involvement of Cl channels in receptor-mediated,
epithelium-dependent relaxation of the airways may be of value in the therapy of airway obstructive diseases such as asthma. Pharmaceuticals may be developed to stimulate Cl
currents and thus
enhance the release of endogenous epithelium-derived relaxing factors.
The exact pathogenesis of cystic fibrosis also remains unclear, and it
has been suggested that there is underlying hyperfunction of airway
smooth muscle in cystic fibrosis (13). Perhaps in humans
where there is insufficient compensation for the loss of the CFTR
channel, impaired release of an epithelium-dependent airway-relaxing
factor may contribute to the pathogenesis of cystic fibrosis. The
present study may therefore have implications for the therapy of one
disease and for better understanding of another.
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ACKNOWLEDGEMENTS |
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This work was supported by an MD/PhD Scholar Award (to C. N. Fortner) and National Heart, Lung, and Blood Institute Grants HL-61974 (to R. J. Paul) and HL-54829 (to R. J. Paul).
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. N. Fortner, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (E-mail: chris.fortner{at}uc.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 4 January 2000; accepted in final form 7 September 2000.
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