EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2

Christopher N. Fortner1, Richard M. Breyer2, and Richard J. Paul1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PGF2alpha 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   ACh concentration-active force for tracheas from wild-type (WT) mice or mice homozygous for a gene-targeted deletion of the PGE2 receptor [EP2(-/-)]. Values are means ± SE for n = 9 wild-type and n = 11 EP2(-/-) tracheas. There were no statistically significant differences. [Acetylcholine], ACh concentration.


                              
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Table 1.   Tracheal segment properties for EP2(-/-) and wild-type mice

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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Fig. 2.   Isometric force recording from EP2(-/-) and wild-type mouse tracheal rings. Typical experimental tracing showing relaxation to substance P (SP) and ATP for wild-type (dotted line) and EP2(-/-) (solid line) tracheas constricted with ACh. Tracheas shown were mounted in the same organ bath and thus exposed to identical conditions. Pharmaceuticals were added at the times indicated by the arrows and remained in the bath until washout with fresh physiological salt solution. Horizontal bar represents 10 min on the time axis. Vertical bar represents 10 mN/mm2 of active force per cross-sectional area.



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Fig. 3.   Peak relaxation to SP or ATP for EP2(-/-) and wild-type mouse tracheas. Relaxation (decrease in force) is expressed as a percentage of the constriction to 10 µM ACh. Values are means ± SE for 9 wild-type and 11 EP2(-/-) tracheas. *P < 0.01.

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.


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Fig. 4.   PGE2 production by EP2(-/-) and wild-type mouse tracheas. PGE2 levels measured by RIA were normalized to tissue weight. Values are means ± SE for n = 4 wild-type and n = 4 EP2(-/-) tracheas. There were no statistically significant differences between wild-type and EP2(-/-) tracheas. Control represents PGE2 production by unstimulated tracheas. ATP (10 µM) significantly increased PGE2 production in both wild-type and EP2(-/-) tracheas compared with control. Pooled, pooled wild-type and EP2(-/-) data analyzed for the effects of SP or ATP vs. control; [PGE2], PGE2 concentration. *P < 0.05.

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.


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Fig. 5.   PGE2 concentration-relaxation for EP2(-/-) and wild-type mouse tracheas. Tracheas were constricted with 10 µM ACh, and force remaining at each concentration of PGE2 is expressed as a percentage of the maximum contraction to 10 µM ACh. Values are means ± SE for n = 9 wild-type and n = 11 EP2(-/-) tracheas. Data for EP2(-/-) do not fit a standard concentration-response curve and are connected with a spline curve only. *P < 0.01.

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 beta -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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Fig. 6.   Isoproterenol concentration-relaxation for EP2(-/-) and wild-type mouse tracheas. Tracheas were constricted with 10 µM ACh, and force remaining at each concentration of isoproterenol is expressed as a percentage of the maximum contraction to 10 µM ACh. Values are means ± SE for 9 wild-type and 11 EP2(-/-) tracheas. There were no statistically significant differences. [Isoproterenol], isoproterenol concentration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank Diane Brockman for expert technical assistance with the PGE2 measurements.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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