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(Chest. 2005;128:3717-3723.)
© 2005 American College of Chest Physicians

Prostaglandin E2 Receptor Selective Agonists E-Prostanoid 2 and E-Prostanoid 4 May Have Therapeutic Effects on Ovalbumin-Induced Bronchoconstriction*

Hiroshi Tanaka, MD; Satoshi Kanako, MD and Shosaku Abe, MD

* From the Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.

Correspondence to: Hiroshi Tanaka, MD, Third Departments of Internal Medicine, Sapporo Medical University school of Medicine, South-1, West-16, Chuo-ku, Sapporo, 060-8543, Japan; e-mail: tanakah{at}sapmed.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The pharmacologic actions of prostaglandin E2 (PGE2) are mediated through specific E-prostanoid (EP)-1, EP-2, EP-3, and EP-4 receptors. In this study, we determined which PGE2 receptor subtype(s) contribute to the prevention of allergen-induced bronchoconstriction.

Methods: We assessed the effects of these receptor agonists in ovalbumin (OA)-sensitized guinea pigs. The prostaglandin E receptor-subtype agonists tested were ONO-DI-004 (EP-1), ONO-AE1–259 (EP-2), ONO-AE-248 (EP-3), ONO-AE1–329 (EP-4), and sulprostone (EP-1 and EP-3) [Ono Pharmaceutical Company; Osaka, Japan]. We treated the animals with either PGE2 or these agonists 15 min before OA challenge and measured respiratory resistance at 15 min, 1 h, and 3 h.

Results: Allergen-induced bronchoconstriction was significantly (p < 0.01) suppressed at doses > 85 nmol/kg of PGE2. The respiratory resistance elevations 15 min after OA challenge were significantly (p < 0.01) suppressed by preadministration of EP-2 and EP-4 agonists, but airway responsiveness to inhaled methacholine did not improve. EP-1, EP-3, or EP-1/EP-3 agonists had no effect on any parameter.

Conclusions: These results suggest that inhibition of OA-induced bronchoconstriction by PGE2 acts through EP-2 and EP-4 receptors.

Key Words: animal model • asthma • prostaglandin E2 • receptor • smooth muscle


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin E2 (PGE2) is a cyclooxygenase product of arachidonate metabolism that relaxes airway smooth muscle.12 PGE2 acts through four different rhodopsin-type receptors (Table 1 ), designated as E-prostanoid (EP)-1, EP-2, EP-3, and EP-4, which are encoded by different genes and expressed differently in each tissue.345 PGE2 counteracts exercise-induced,6 allergen-induced,78 and aspirin-induced bronchoconstriction.910 Inhaled PGE2 prevents allergen-induced asthmatic responses when administered immediately before allergen challenge,11 while having no effect on baseline FEV1 or on methacholine hyperreactivity.12 However, PGE2 can both relax and constrict airway smooth muscle in vitro and ex vivo.1113 This paradox was explained when it was recognized that PGE2 could simultaneously stimulate four different receptors, which are coupled to different intracellular signal transduction pathways. In vascular smooth muscle, PGE2 is generally found to be a dilator through EP-2 or EP-4 receptors and a constrictor through EP-1 or EP-3 receptors.14 Both PGE2-elicited contraction and the spontaneous tone of guinea pig tracheal smooth muscle were reported to be mediated through the activation of the EP-1 receptor.15 Tilley et al14 reported that the activation of EP-1/EP-3 receptors induced airway constriction, whereas PGE2-induced bronchodilation resulted from activation of the EP-2 receptor in airway smooth muscle. In this study, we attempted to identify which EP receptor is involved in the protective effects of PGE2 on ovalbumin (OA)-induced bronchoconstriction, using a specific agonist for each EP receptor in OA-sensitized guinea pigs.


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  		Table 1.. Receptors for PGE2*

 

   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Drugs
Male Hartley guinea pigs weighing 250 to 300 g (Nihon SLC; Shizuoka, Japan) were allowed bottled water and commercial chow ad libitum. PGE2 was obtained from Sigma Chemical Company (St. Louis, MO). ONO-DI-004 (EP-1 agonist), ONO-AE1–259 (EP-2 agonist), ONO-AE-248 (EP-3 agonist), ONO-AE1–329 (EP-4 agonist), and sulprostone (EP-1 and EP-3 agonists)5 were gifts kindly provided by the Ono Pharmaceutical Company Ltd. (Osaka, Japan). The specificity of the respective EP agonists was analyzed by measuring the binding affinity of the agonists to the respective EPs expressed in Chinese hamster ovary cells.34

Study Design
The guinea pigs were placed in polyacrylamide boxes (28 x 28 x 20 cm) and sensitized by using aerosolized 1% OA solution delivered via an ultrasonic nebulizer (NE-U10B; Omron; Tokyo, Japan). Sensitization was performed for 10 min once a week for 4 weeks. We have previously reported these procedures.1617 An OA challenge test was then performed, and OA-induced bronchoconstriction was observed in all animals. All experiments and procedures were approved by the Laboratory Animal Center of Sapporo Medical University.

Protocol of Experiment 1
As shown in Figure 1 , we allocated the guinea pigs into two groups: a vehicle group (physiologic saline solution, 0.09 NaCl weight/volume in sterile water, subcutaneously) and a PGE2 group (1, 3, 10, and 50 µg/kg, subcutaneously). Stock solution of PGE2 was prepared at a concentration of 2 mg/mL by dissolving 10 mg of PGE2 in 5.0 mL of 100% ethanol. This solution was stored at – 70°C. Immediately before use, the PGE2 was diluted with physiologic saline solution. Treatment was administered 15 min before OA challenge, and specific airway resistance (sRaw) was measured before and 15 min, 1 h, and 3 h after OA challenge. The vehicle control group consisted of 10 animals, and each treatment group consisted of seven animals.



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  		Figure 1.. Protocol of experiment 1.

 
Protocol of Experiment 2
OA-sensitized animals were classified into six groups as shown in Figure 2 : the vehicle group (dimethylsulfoxide [DMSO], subcutaneously); the EP-1 receptor agonist group (ONO-DI-004 dissolved in methyl cellulose at doses of 20 nmol/kg and 200 nmol/kg body weight, po); the EP-2 receptor agonist group (ONO-AE1–259 dissolved in DMSO at doses of 20 nmol/kg and 200 nmol/kg body weight, subcutaneously); the EP-3 receptor agonist group (ONO-AE-248 dissolved in DMSO at doses of 20 nmol/kg and 200 nmol/kg body weight, subcutaneously); the EP-4 receptor agonist group (ONO-AE1–329 dissolved in DMSO at doses of 2 nmol/kg and 20 nmol/kg of body weight, subcutaneously); and the EP-1 and EP-3 receptor agonist group (sulprostone dissolved in DMSO at doses of 20 nmol/kg and 200 nmol/kg body weight, subcutaneously). Drugs were administered 15 min before OA challenge, and SRaw was measured before and 15 min and 1 h after OA challenge. OA-induced bronchoconstriction in OA-sensitized guinea pigs was not affected by the oral low dose of methyl cellulose (data not shown). The vehicle control group consisted of 10 animals, and each treatment group consisted of 7 animals.



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  		Figure 2.. Protocol of experiment 2.

 
Protocol of Experiment 3
To measure the concentration of airway PGE2, we performed BAL. OA-sensitized animals were classified into five groups similar to that shown in Figure 2: the vehicle group (DMSO, subcutaneously), the EP-1 receptor agonist group (ONO-DI-004, 200 nmol/kg po), the EP-2 receptor agonist group (ONO-AE1–259, 200 nmol/kg subcutaneously), the EP-3 receptor agonist group (ONO-AE-248, 200 nmol/kg subcutaneously), and the EP-4 receptor agonist group (ONO-AE1–329, 20 nmol/kg subcutaneously) groups. BAL was performed 15 min after OA challenge.

Measurement of Airway Function
sRaw was measured using respective sensors of air flow equipped at the front and rear chambers according to the method described by Pennock et al,17 and by us.1617 Briefly, an awake guinea pig was positioned with its neck extending through the partition of a two-chamber, whole-body plethysmograph. Each chamber was fitted with identical wire screen pneumotachographs and identical differential pressure transducers (model DP-45; Validyne Engineering Corporation; Northridge, CA). These transducers were connected to a respiratory analyzer (Pulmos-I; M.I.P.S.; Osaka, Japan), and a personal computer was used for the on-line breath-to-breath measurement of sRaw.

Airway Responsiveness to Methacholine
Airway responsiveness (AR) was evaluated by the airway response to methacholine as previously described by us.1516 Methacholine aerosols were generated by an ultrasonic nebulizer (model T-10; Sineo Industries; Saitama, Japan), with a flow rate of 0.8 L/min delivered into the chamber. Methacholine solutions were prepared in concentrations of 0.0078, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, and 0.1 mg/mL. After each inhalation for 1 min, sRaw was measured for 1 min. Inhalation was repeated with aerosols of increasing concentrations until sRaw increased to more than twice baseline. AR was evaluated by the concentration of methacholine required to increase sRaw to twice the baseline level.

BAL
After the animal received a lethal dose of pentobarbital sodium (100 mg/kg intraperitoneally), the chest was opened and both lungs were lavaged by instillation and withdrawal of four aliquots of 1 mL of sterile pathogen-free saline solution. BAL fluid was placed on ice and centrifuged at 400g for 5 min at 4°C. Supernatants were decanted and frozen at – 80°C for subsequent use. Levels of PGE2 were determined by radioimmunoassay (Prostaglandin E2 [125I] RIA Kit; PerkinElmer Life Sciences; Boston MA). The kit was able to detect concentrations of PGE2 as low as 0.1 pg/mL.

Statistical Analysis
Results are expressed as mean and SEM, or median and 10th to 90th percentiles. Statistical analysis was performed using a two-factor, repeated-measures analysis of variance. When differences were significant, the Fisher least significant difference test for multiple comparison was applied. Values were considered significantly different at p < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PGE2 on OA-Induced Bronchoconstriction
The results of sRaw measured before and 15 min, 1 h, and 3 h after OA challenge are shown in Figure 3 . PGE2 significantly reduced the sRaw at 15 min in the group receiving > 3 µg/kg of PGE2 compared with sham control animals, and sRaw 1 h after OA challenge was significantly decreased only in the group receiving 50 µg/kg of PGE2. There was no significant difference between the values obtained from different treatment doses at 3 h after OA challenge among the groups.



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  		Figure 3.. Effect of PGE2 on OA-induced bronchoconstriction. PGE2 significantly reduced sRaw 15 min after OA challenge in a dose-dependent manner.

 
Effect of Different EP Receptor Antagonist
The baseline data of sRaw before OA challenge were from 1.6 to 2.4 cm H2O/mL/s, and there were no significant differences among the treatment groups. Both ONO-AE1–259-01 and ONO-AE1–329 suppressed OA-induced bronchoconstriction, and the extent of the inhibition was similar to that achieved by PGE2 (Fig 4 ). In contrast, ONO-DI-004, ONO-AE-248, and sulprostone did not inhibit the bronchoconstriction caused by OA challenge. The changes in AR to methacholine ({Delta} of log concentration of methacholine required to increase sRaw to twice the baseline level) on treatment with vehicle and different EP receptor antagonists were as follows: vehicle (0.09 ± 0.24); ONO-DI-004 (20 nmol, 0.11 ± 0.16; 200 nmol, 0.45 ± 0.18); ONO-AE1–259 (20 nmol, 0.09 ± 0.14; 200 nmol, 0.06 ± 0.17); ONO-AE-248 (20 nmol, 0.12 ± 0.15; 200 nmol, 0.07 ± 0.17); ONO-AE1–329 (2 nmol, 0.08 ± 0.10; 20 nmol, 0.11 ± 0.14); and sulprostone (20 nmol, 0.06 ± 0.25; 200 nmol, 0.11 ± 0.22). There were no significant differences among these groups. As shown in Figure 5 , there were no significant differences in PGE2 levels in BAL fluid among the five treatment groups: vehicle, ONO-DI-004, ONO-AE1–259, ONO-AE-248, and ONO-AE1–329.



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  		Figure 4.. Effect of each EP receptor antagonist on OA-induced bronchoconstriction. Both ONO-AE1–259-01 (EP-2 agonist) and ONO-AE1–329 (EP-4 agonist) significantly suppress (analysis of variance) the OA-induced bronchoconstriction as compared with sham control animals. ONO-DI-004 (EP-1 agonist), ONO-AE-248 (EP-3 agonist), and sulprostone (an EP-1 and EP-3 agonist) produce no suppression.

 


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  		Figure 5.. BAL fluid PGE2 levels in each treatment group. Each group consists of seven guinea pigs. No significant differences exist among vehicle, ONO-DI-004 (EP-1 agonist), ONO-AE1–259 (EP-2 agonist), ONO-AE-248 (EP-3 agonist), and ONO-AE1–329 (EP-4 agonist). The rectangle includes the range from the 25th to 75th percentiles, the horizontal line indicates the median, and the vertical line indicates the range from the 10th to 90th percentiles.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrated that inhibition of OA-induced bronchoconstriction by PGE2 occurs through EP-2 and EP-4 receptors in a sensitized guinea pig model. Our results also demonstrated for the first time that both EP-2– and EP-4–selective agonists inhibited the OA-induced bronchoconstriction when administered immediately before OA challenge, but these treatments did not affect AR. Fortner et al,18 using mice homozygous for a gene targeted deletion of the EP-2 receptor, reported that EP-2 receptors were of primary importance in the airway relaxation to PGE2 and that relaxation to adenosine triphosphate was mediated through PGE2 acting on EP-2 receptors. Using a knockout mouse model, Tilley et al14 found that PGE2-induced bronchodilation resulted from activation of the EP-2 receptor on airway smooth muscle, whereas activation of EP-1/EP-3 receptors induced airway constriction. Our results are consistent with these previous reports. Activation of EP-1 receptors increases intracellular Ca2+, and EP-3 couples to inhibitory guanosine triphosphate (GTP)-binding protein with a decrease in cyclic adenosine monophosphate (cAMP). However, EP-2 and EP-4 are coupled to stimulatory GTP-binding protein, activating adenylyl cyclase and increasing cAMP, a signal known to relax airway smooth muscle. In total, these findings indicate that the bronchoprotective effects of PGE2 may occur through stimulation of EP-2 and EP-4 receptors but not through EP-1 and EP-3 receptors.

The effects of PGE2-induced smooth-muscle relaxation in vitro are sometimes inconsistent,1113 probably due to simultaneous stimulation of different prostaglandin-like receptors with inhibitory and stimulatory activity on intrinsic tone. We suppose that PGE2 inhibition of the bronchoconstriction may occur at levels consistent with an interaction of the PGE2 receptors. The strong bronchoprotective effects of PGE2 have not been exploited for asthma therapy partly because of its adverse effects of cough and retrosternal burning.20 Additionally, PGE2 can stimulate intrapulmonary C fibers and to a lesser extent rapidly adapting irritant receptors.21 Our results suggest that EP-2 or EP-4 receptor agonists might have better therapeutic potential rather than PGE2, provided EP-2 or EP-4 receptor agonists do not have adverse effects.

This protective action of PGE2 might not only result from a direct effect on airway smooth-muscle relaxation but also by inhibition of many inflammatory processes, including mast-cell degranulation, leukotriene B4 production by alveolar macrophages, and eosinophil activation.782223 PGE2 inhalation suppresses mast-cell release of prostaglandin metabolites, resulting in significantly lower concentrations of prostaglandin D2 after antigen stimulation.23 However, in the preliminary studies, the EP-2 and EP-4 agonists used in our experiments did not suppress the release of prostaglandin D2 from mast cells, and these agonists had no effect on methacholine-induced airway responsiveness. PGE2 levels in BAL fluid did not differ among the treatment groups, suggesting that none of the EP agonists act to release PGE2 from any of the possible cell types. Mechanisms of the bronchoprotective actions of EP-2 and EP-4 require further examination.

In conclusion, the inhibition of airway smooth-muscle contraction by PGE2 acts through EP-2 and EP-4 receptors in OA-sensitized guinea pigs. Although PGE2 might be of therapeutic benefit, PGE2 itself has the potential to cause the adverse effects of cough and retrosternal burning when inhaled by humans. Moreover, PGE2 also stimulates other prostaglandin-like receptors having inhibitory and stimulatory activity on the intrinsic tone of the airway smooth muscle. Thus, it is feasible to suggest that EP-2 or EP-4 receptor agonists, if free of adverse effects on human airways, may have therapeutic benefit instead of inhaled PGE2.


   Footnotes
 
Abbreviations: AR = airway responsiveness; cAMP = cyclic adenosine monophosphate; DMSO = dimethylsulfoxide; EP = E-prostanoid; GTP = guanosine triphosphate; OA = ovalbumin; PGE2 = prostaglandin E2; sRaw = specific airway resistance

Received for publication November 19, 2004. Accepted for publication May 24, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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