1 Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576; and 2 Division of Nephrology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-2372
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ABSTRACT |
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Substance P (SP) and ATP evoke
transient, epithelium-dependent relaxation of constricted mouse
tracheal smooth muscle. Relaxation to either SP or ATP is blocked by
indomethacin, but the specific eicosanoid(s) involved have not been
definitively identified. SP and ATP are reported to release
PGE2 from airway epithelium in other species, suggesting
PGE2 as a likely mediator in epithelium-dependent airway
relaxation. Using mice homozygous for a gene-targeted deletion of the
EP2 receptor [EP2(/
)], one of the
PGE2 receptors, we tested the hypothesis that
PGE2 is the primary mediator of relaxation to SP or ATP.
Relaxation in response to SP or ATP was significantly reduced in
tracheas from EP2(
/
) mice. There were no differences between EP2(
/
) and wild-type tracheas in their physical
dimensions, contraction to ACh, or relaxation to isoproterenol, thus
ruling out any general alterations of smooth muscle function. There
were also no differences between EP2(
/
) and wild-type
tracheas in basal or stimulated PGE2 production. Exogenous
PGE2 produced significantly less relaxation in
EP2(
/
) tracheas compared with the wild type. Taken
together, this experimental evidence supports the following two
conclusions: EP2 receptors are of primary importance in
airway relaxation to PGE2 and relaxation to SP or ATP is
mediated through PGE2 acting on EP2 receptors.
smooth muscle; airway; adenosine 5'-triphosphate; mice; mouse; prostaglandin E2
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INTRODUCTION |
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THE AIRWAY EPITHELIUM is an important source of relaxing factors that may prevent or lessen the severity of airway constriction. Damage to the epithelium increases airway hyperreactivity (14), and epithelial shedding is a characteristic of asthma (12). We have recently reported that substance P (SP) and ATP evoke epithelium-dependent relaxation of constricted mouse trachea (10). Denuding the tracheas of epithelium reduces the relaxation to SP or ATP, suggesting the loss of an epithelium-derived relaxing factor or factors. Identifying this factor could not only provide insight into specific mechanisms by which airway epithelium protects against airway constriction, it could also suggest potential therapeutic targets for conditions where a decrease in epithelial cell function may lead to airway hyperreactivity.
Relaxation to SP or ATP is blocked by indomethacin (10,
13), suggesting the possible involvement of prostanoid mediators in those responses. SP has been shown to release PGE2 from
rat airways (7), and ATP releases PGE2 and
PGF2 from rabbit airway epithelium (1).
These previous reports suggest PGE2 as a likely candidate
mediator in the response to SP or ATP. The challenge lies in testing
the hypothesis that PGE2 is the essential mediator in the
relaxation to SP or ATP. One would need to show not only that SP or ATP
releases PGE2 but that blocking the release or action of
PGE2 specifically would inhibit the relaxation to SP or
ATP. The pharmacological tools to test this hypothesis are limited.
There are no specific PGE synthase inhibitors, and receptor antagonists
are not currently available for all of the four PGE2 receptor (EP) subtypes (4).
To test the hypothesis that PGE2 mediates SP- and
ATP-evoked airway relaxation, we used mice in which the cAMP-coupled
EP2 receptor was eliminated through targeted gene
disruption. Each of the four PGE2 receptors has been
disrupted in separate mouse models (reviewed in Ref.
3). The EP2-deficient
[EP2(/
)] mice have a documented defect in
PGE2-mediated relaxation of a smooth muscle tissue, and the
dilator effects of the EP2 receptor in the vasculature
predominates over the effects of activation of other receptor subtypes
in vascular smooth muscle beds (11, 21). Thus tracheas
from EP2(
/
) mice were used to test airway responses to
exogenous PGE2 and to test whether PGE2 is the
primary mediator of the epithelium-dependent relaxation to SP or ATP.
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MATERIALS AND METHODS |
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EP2-deficient mice.
Targeted deletion of the EP2 receptor was previously
reported in detail (11). Pairs of F2 C57/B6 × 129 mice homozygous for deletion of the EP2 receptor
[EP2(/
)] and littermate controls (wild type) aged
10-14 wk were selected for experimentation.
Tracheal tissue preparation. Mice were killed by CO2 asphyxiation, and the trachea was removed and prepared as previously described (10). Once mounted in the experimental apparatus, tracheas were bathed with a 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 and 5% CO2.
Pharmaceuticals. Unless otherwise noted, chemicals were obtained from Sigma (St. Louis, MO). ACh, SP, ATP, and isoproterenol were dissolved in distilled H2O to produce concentrated stocks. PGE2 (Cayman Chemical, Ann Arbor, MI) was dissolved in ethanol to produce 1 mM stock. Subsequent dilutions of PGE2 were in water. When added to the buffer solution, the final concentration of ethanol was <0.3% vol/vol. In vehicle control experiments, this concentration of ethanol had no effect on airway constriction or relaxation.
Force measurements in tracheal ring preparations.
Tracheal rings with the epithelial layer undisturbed were mounted
isometrically to force transducers as previously described in detail
(10). Paired tracheas from EP2(/
) and
wild-type mice were mounted in the same chamber, bathed by the same
buffer solution, and thus exposed to identical concentrations of
constricting or relaxing agents. The n values reported in
this study are the number of mice, with one tracheal segment studied
from each mouse.
Measurements of PGE2 production. After the isometric force studies were completed, tracheas were moved to small holders fashioned from 4.9-mil wire to facilitate collection of PGE2 samples. Samples were collected in 400-µl volumes and bubbled through a 30-gauge needle. Three collection periods consisting of 10 min under unstimulated conditions, followed by 10 min in the presence of 10 nM SP, then 10 min in the presence of 10 µM ATP, and 10 min resting in a separate 100-ml reservoir of PSS were performed for each tissue studied for PGE2 production. PGE2 levels were measured by RIA as previously described in detail (20).
Fitting concentration-response curves. A logistic equation was fit to the mean responses at each concentration of ACh, isoproterenol, or PGE2 using an iterative, nonlinear least squares fit. Calculations were performed with Origin data analysis software (Microcal Software, Northampton, MA).
Data analysis. Data are expressed as means ± SE. The n value is the number of mice used. Standard ANOVA was used, with P < 0.05 taken as evidence of statistical significance. For PGE2 measurements, this was a two-way repeated-measures ANOVA. Bonferroni's method was used to verify the significance level of differences. Statistical analysis was performed with SigmaStat (SPSS, Chicago, IL).
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RESULTS |
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Contraction to ACh.
To determine if lack of EP2 receptors affected airway
contractility to cholinergic stimulation, cumulative
concentration-response curves to ACh were generated for wild-type and
EP2(/
) tracheas (Fig. 1).
There were no differences in peak active force between wild-type and
EP2(
/
) tracheas. Sensitivity to ACh was also not significantly different between wild-type (EC50 = 4.7 ± 0.7 µM, n = 9) and EP2(
/
)
(EC50 = 5.4 ± 0.9 µM, n = 11)
tracheas. At the end of all experiments, tracheal segments were weighed
and measured, since tissue size contributes to the maximum force
generated. There were no significant differences in the physical
properties of tracheal segments from EP2(
/
) mice
compared with wild-type mice (Table 1).
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Relaxation to SP or ATP.
For studies of airway relaxation, tracheas were constricted with 10 µM ACh. On the basis of the ACh concentration-response curves (Fig.
1), 10 µM ACh is approximately the ED80 for ACh in both
wild-type and EP2(/
) tracheas. Contractions to 10 µM
ACh were stable within 10 min of adding ACh to the tissue bath. A typical force recording is shown in Fig.
2. After stable contractions to 10 µM
ACh were observed, 10 nM SP induced transient relaxation of constricted
tracheas. The full magnitude of contraction was recovered within 15 min, at which time 10 µM ATP was added, leading to a second transient
relaxation. Peak relaxation to SP or ATP was compared between wild-type
and EP2(
/
) tracheas; data from these types of
experiments are summarized in Fig. 3.
Relaxation to SP was significantly reduced in EP2(
/
)
tracheas, as was relaxation to ATP.
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PGE2 measurements.
An alternative hypothesis to explain the observed defect in relaxation
to SP or ATP is that EP2(/
) tracheas might release less
PGE2 in response to SP or ATP rather than the differences being due to a defect in the response to PGE2. This
hypothesis was tested by measuring PGE2 production by
wild-type and EP2(
/
) tracheas. Under unstimulated
conditions, all tracheas produced PGE2, with no differences
between the wild type and EP2(
/
) (Fig. 4). ATP significantly increased the
production of PGE2 (P = 0.01) in both
wild-type and EP2(
/
) tracheas. SP also increased
PGE2 production; however, this increase was not
statistically significant. There were no differences between wild-type
and EP2(
/
) production of PGE2 in the
presence of SP or ATP.
|
Relaxation to exogenous PGE2.
The impaired relaxation to SP or ATP in EP2(/
) tracheas
could also be a result of impaired relaxation to PGE2
released in response to SP or ATP. We tested this hypothesis by
measuring cumulative concentration-response curves for exogenous
PGE2 treatment of wild-type and EP2(
/
)
tracheas constricted with 10 µM ACh (Fig.
5). Relaxation to PGE2 was
significantly impaired in EP2(
/
) tracheas for
concentrations of 1 nM PGE2 or greater. At concentrations >100 nM PGE2, EP2(
/
) tracheas exhibited a
constriction response to PGE2.
|
Relaxation to isoproterenol.
EP2 receptors are reported to signal through adenylate
cyclase (9). The observed defect in relaxation to
PGE2 could potentially be a result of a more general defect
in cAMP-dependent relaxation. To test this hypothesis, a second
receptor-mediated, cAMP-dependent smooth muscle relaxant was
studied. Cumulative concentration-response curves to the
-adrenergic agonist isoproterenol were generated for wild-type and
EP2(
/
) tracheas (Fig. 6).
There were no significant differences in the isoproterenol
concentration-response curves between wild-type and
EP2(
/
) tracheas. Peak relaxation to isoproterenol was
significantly less than the peak relaxation to PGE2
observed in tracheas from wild-type mice but was consistent with
previously published relaxation values for isoproterenol in mouse
trachea (15).
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DISCUSSION |
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This study was designed to test the hypothesis that PGE2 is the essential mediator of epithelium-dependent airway relaxation to SP or ATP. Previous studies have shown that PGE2 is released from airway epithelium in response to SP or ATP (1, 7), suggesting PGE2 as a likely mediator. To test whether the responses depend specifically on PGE2, it is necessary not only to demonstrate the release of PGE2 but also to specifically block PGE2 function. Although indomethacin will block PGE2 production by inhibiting cyclooxygenase, it also blocks the production of many other eicosanoids, several of which could be key relaxing factors. There are no published reports of any specific inhibitors of PGE synthase nor are there any reported genetic models of PGE synthase deficiency. Another approach to showing a dependence on PGE2 would be studies with antagonists to block the action of PGE2 specifically. This approach is complicated by the presence of four PGE2 receptor subtypes (6) and a lack of specific antagonists for the EP2 and the EP3 receptor subtypes (for review see Ref. 4). However, each EP receptor subtype has been ablated in separate mouse models, making the tools available to test whether relaxation to SP or ATP depends on PGE2.
Of the four EP receptors, two mediate their signals through second
messengers that would tend to constrict smooth muscle. EP1
signals through an increase in intracellular calcium (8). Mice deficient in EP1 show impaired contraction of vascular
smooth muscle, supporting the idea that EP1 mediates
constriction of smooth muscle (2). EP3 signals
through a decrease in cAMP, and mice deficient in EP3 show
impaired febrile responses (19). The remaining two EP
receptors signal through an increase in cAMP, a signal known to relax
smooth muscle. Mice deficient in EP4 show defective closure
of the ductus arteriosus at birth (16, 17). The
EP2(/
) mice exhibit a documented defect in smooth
muscle relaxation (11), suggesting EP2 as a
logical starting point for investigating PGE2-mediated
relaxation of airway smooth muscle.
For EP2(/
) tracheas to be a suitable tool for
investigating airway physiology, it was important to determine if there
were any gross differences in airway structure or contractile function. EP2(
/
) tracheas did not have different physical
dimensions from the wild type nor did lack of EP2 receptors
affect tracheal constriction to ACh. The lack of differences in
contractility not only supports EP2(
/
) mouse tracheas
as a suitable airway model, it facilitates comparison between
EP2(
/
) and wild-type tracheas for relaxation to SP,
ATP, PGE2, or isoproterenol.
The responses to SP or ATP have been previously established as
epithelium-dependent phenomena (10) and may be important for preventing or lessening the severity of airway constriction. An
exciting finding from the current study was that relaxation to SP or
ATP was significantly impaired in EP2(/
) tracheas. This
supports the hypothesis that PGE2, signaling through
EP2 receptors, is the primary mediator of relaxation to SP
or ATP.
One alternative hypothesis is that PGE2 production may be
reduced in EP2(/
) tracheas. Measurements of
PGE2 production revealed that EP2(
/
)
tracheas showed no defect in PGE2 production compared with
the wild type in either unstimulated conditions or in response to SP or
ATP stimulation. Epithelial cell PGE2 production was not
specifically assessed in the tracheal ring preparation, and thus
differences in tissue-specific PGE2 production cannot be ruled out and may be worth further study. The results may also be
explained by the hypothesis that the defect in epithelium-dependent relaxation is a result of EP2(
/
) tracheas not relaxing
as effectively to the PGE2 released in response to SP or
ATP. The finding that relaxation to exogenous PGE2 is
significantly impaired in EP2(
/
) tracheas supports this
hypothesis. These data are also consistent with in vivo studies
demonstrating the loss of PGE2-mediated relaxation of
muscarinic contractions in the airways of EP2-deficient
mice (18), confirming an important role for the
EP2 receptor in airway relaxation to PGE2.
It is interesting that EP2(/
) tracheas showed a slight
relaxation to PGE2. This relaxation may be mediated through
one of the other EP receptors, possibly EP4, which is also
expressed in airways (5). At higher concentrations of
PGE2, the constriction to PGE2 in
EP2(
/
) tracheas occurred in concentration-dependent steps when PGE2 was added to the bath. These contractions
may be caused by EP1 or EP3 receptors, which
have been previously shown to mediate contraction of vascular smooth
muscle from wild-type and EP2(
/
) mice
(11). These receptors may also be functioning in wild-type
mouse airways; however, the EP2 receptor dominates the
response to PGE2, resulting in relaxation. This would be
similar to the predominance of the EP2 receptor-mediated
effects observed in vascular smooth muscle beds (21).
Another alternative hypothesis is that disrupting the EP2
receptor may have affected relaxation pathways in a nonspecific manner.
If this were the case, the impaired relaxation to SP, ATP, or
PGE2 could all be the result of a generalized defect in relaxation of airway smooth muscle rather than a specific deficiency in
EP2 receptor-mediated signaling. The defect could be at the level of Gs, adenylate cyclase, or even in the smooth
muscle response to increased cAMP. This alternative hypothesis was
tested using isoproterenol, another agonist that mediates smooth muscle
relaxation through Gs-coupled receptors and increased cAMP.
Normal relaxation to isoproterenol in EP2(/
) tracheas
suggests that the defect in relaxation to PGE2 is due
specifically to lack of EP2 receptors rather than a
nonspecific defect downstream of the receptor.
In summary, this study presents direct evidence that PGE2
mediates airway relaxation through EP2 receptors. When
EP2 receptors are ablated through targeted gene deletion,
relaxation to PGE2 is significantly impaired. Relaxation to
SP or ATP is similarly impaired in tracheas from EP2(/
)
mice. The defect in relaxation to SP or ATP in EP2(
/
)
tracheas is specifically a result of impaired relaxation to
PGE2, mediated through EP2 receptors. It is not
the result of impaired production of PGE2 in response to SP
or ATP. Thus strong experimental evidence is provided to support the
role of PGE2 as the epithelium-derived relaxing factor
released from airway epithelium in response to SP or ATP.
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ACKNOWLEDGEMENTS |
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We thank Diane Brockman for expert technical assistance with the PGE2 measurements.
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FOOTNOTES |
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This work was supported by MD/PhD Scholar Award (C. N. Fortner) and National Institutes of Health Grants GM-15431 (R. M. Breyer), DK-46205 (R. M. Breyer), HL-61974 (R. J. Paul), and HL-54829 (R. J. Paul).
Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576. (E-mail: richard.paul{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 22 June 2000; accepted in final form 27 February 2001.
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