Effect of inhaled prostaglandin E 2 on methacholine and leukotriene D 4 airway responsiveness in asthmatic subjects

Previous studies in asthmatics have demonstrated that the endogenous release of inhibitory prostaglandins limits the bronchoconstrictor response to repeated challenges with exercise and histamine, and that inhaled prostaglandin (PG) E2 attenuates allergen-induced asthmatic responses and exercise bronchoconstriction in asthmatics. Inhaled PGE2 does not significantly attenuate methacholine airway responsiveness. These results, taken together, indicate that inhaled PGE2 attenuates the bronchoconstriction caused by stimuli, such as allergen and exercise, that result in bronchoconstriction through cysteinyl leukotriene (LT) release. The purpose of this study was to determine whether inhaled PGE2 could selectively attenuate LTD4-induced bronchoconstriction in seven stable asthmatic subjects. Each subject was studied on four different study days. On two occasions the subjects inhaled 100 mg PGE2, 30 mins before a methacholine, or LTD4 challenge test. On the other two study days, the subjects were pretreated with its diluent. Results were expressed as the provocation concentration causing a 20% fall in forced expiratory volume in 1 s (FEV1) (PC20). PGE2 pretreatment significantly increased the LTD4 PC20, but not the methacholine PC20. The mean LTD4 PC20 increased from 2.00 mg/mL (%SEM 1.65) after diluent pretreatment to 3.01 mg/mL (%SEM 1.64) after PGE2 pretreatment (P=0.008). The mean methacholine PC20 was 1.28 mg/mL (%SEM 1.68) after diluent pretreatment and 1.62 mg/mL (%SEM 1.46) after PGE2 pretreatment (P=0.28). These results suggest that PGE2 partially attenuates LTD4-induced bronchoconstriction; however, the magnitude of the effect is unlikely to account for its attenuation of exercise and allergen-induced bronchoconstriction.

Previous studies in asthmatics have demonstrated that the endogenous release of inhibitory prostaglandins limits the bronchoconstrictor response to repeated challenges with exercise and histamine, and that inhaled prostaglandin (PG) E 2 attenuates allergen-induced asthmatic responses and exercise bronchoconstriction in asthmatics.Inhaled PGE 2 does not significantly attenuate methacholine airway responsiveness.These results, taken together, indicate that inhaled PGE 2 attenuates the bronchoconstriction caused by stimuli, such as allergen and exercise, that result in bronchoconstriction through cysteinyl leukotriene (LT) release.The purpose of this study was to determine whether inhaled PGE 2 could selectively attenuate LTD 4 -induced bronchoconstriction in seven stable asthmatic subjects.Each subject was studied on four different study days.On two occasions the subjects inhaled 100 mg PGE 2 , 30 mins before a methacholine, or LTD 4 challenge test.On the other two study days, the subjects were pretreated with its diluent.Results were expressed as the provocation concentration causing a 20% fall in forced expiratory volume in 1 s (FEV 1 ) (PC 20 ).PGE 2 pretreatment significantly increased the LTD 4 PC 20 , but not the methacholine PC 20 .The mean LTD 4 PC 20 increased from 2.00 mg/mL (%SEM 1.65) after diluent pretreatment to 3.01 mg/mL (%SEM 1.64) after PGE 2 pretreatment (P=0.008).The mean methacholine PC 20 was 1.28 mg/mL (%SEM 1.68) after diluent pretreatment and 1.62 mg/mL (%SEM 1.46) after PGE 2 pretreatment (P=0.28).These results suggest that PGE 2 partially attenuates LTD 4 -induced bronchoconstriction; however, the magnitude of the effect is unlikely to account for its attenuation of exercise and allergen-induced bronchoconstriction.
Inhaled LTD 4 is a potent bronchoconstrictor mediator of human airways (8,9).Exercise-and allergen-induced bronchoconstriction can be largely abolished by pretreatment with LTD 4 receptor antagonists (10)(11)(12) or synthetase inhibitors (13,14), thereby implicating LTD 4 as an important mediator in causing exercise-and allergen-induced bronchoconstriction.The fact that inhaled PGE 2 selectively inhibits bronchoconstrictor responses to these stimuli caused by LTD 4 , but not to methacholine-induced bronchoconstriction, raises the possibility that PGE 2 can selectively antagonize LTD 4 -induced bronchoconstriction.The purpose of this study, therefore, was to evaluate whether inhaled PGE 2 , administered in doses known to attenuate exercise-and allergen-induced bronchoconstriction, also attenuates LTD 4 -or methacholineinduced airway responsiveness in stable asthmatic subjects.

Subjects:
Seven stable asthmatic subjects (five females, two males), aged between 19 and 42 years, were studied when their asthma was controlled by the as-required use of inhaled beta 2 -agonist alone.The subjects had no exacerbations of asthma for at least eight weeks before the study, and baseline forced expiratory volume in 1 s (FEV 1 ) was 80% predicted normal (15) in all subjects on each study day.Subjects were instructed to withhold use of inhaled bronchodilators at least 8 h before challenges.All subjects were atopic as demonstrated by at least one positive skin test to a battery of 16 common allergens.The project was approved by the Ethics Committee of McMaster University Medical Centre, and each subject gave written informed consent before taking part.Study design: All subjects attended the laboratory for five study periods.The first period was a screening day during which subjects' characteristics, including methacholine airway responsiveness, were documented.During the next four study periods baseline spirometry was measured and subjects were pretreated with either inhaled PGE 2 or its diluent.Spirometry was repeated 5 mins and 30 mins after the diluent or PGE 2 pretreatment, followed immediately by an LTD 4 or methacholine inhalation test.In an effort to blind the investigator doing the methacholine or LTD 4 challenges, a different investigator delivered the PGE 2 and diluent pretreatments in a different room from that used for the inhalation challenge procedures.The study used a single blinded, diluent controlled, crossover design.All spirometric measurements were made using a 14 L water spirometer (Warren E Collins Inc, Massachusetts).PGE 2 or diluent pretreatment: The PGE 2 pretreatment was as previously described ( 5) by this laboratory.PGE 2 stock solution (2 mg/mL) was prepared by diluting dry powder (Sigma, Missouri) in ethanol and stored at -70°C.One millilitre of the stock solution of PGE 2 was diluted with 0.2 mL 0.9% saline and delivered using a breath-activated dosimeter (PK Morgan, Gillingham, United Kingdom) set to produce an output of 10 mg (0.006 mL PGE 2 ) per breath.Subjects were instructed to take 10 deep breaths of the aerosolized solution, for a total dose of 100 mg.The diluent was prepared by diluting 1 mL of ethanol in 0.2 mL saline.Methacholine inhalation test: Methacholine inhalation was performed as previously described (16).Doubling concentrations of methacholine were inhaled from a Wright nebulizer (Roxon) beginning with a concentration of 0.03 mg/mL for periods of 2 mins.Following each inhalation period, FEV 1 was measured at 30 s, 1.5 mins, 3 mins and then every 2 mins, if necessary, until the lowest value was obtained.Once a fall in FEV 1 of 20% or greater occurred, the test was terminated and the concentration of methacholine required to produce a fall in FEV 1 of 20% was calculated, and expressed as the provocative concentration causing a 20% fall in FEV 1 (methacholine PC 20 ).After the test, two puffs of salbutamol (200 mg) were given to reverse the bronchoconstriction.LTD 4 inhalation test: The LTD 4 inhalation was done as previously described (4).Subjects inhaled 10 breaths of increasing doubling concentrations of LTD 4 , from 0.025 to 50 mg/mL, at intervals of 5 mins, from a breath-activated dosimeter (PK Morgan) set to produce an output of 10 mg.Stock solutions of LTD 4 diluted in dH 2 O (1 mg/mL) (Merck PGE 2 en inhalation pouvait de façon sélective atténuer la bronchoconstriction induite par les LTD 4 chez sept sujets asthmatiques stables.Chaque sujet a été étudié lors de quatre jours d'étude différents.À deux occasions, ils ont inhalé 100 mg de PGE 2 , 30 minutes avant de subir un test de provocation à la méthacholine, ou aux LTD 4 .Pendant les deux autres jours d'étude, les sujets ont été prétraités avec le diluant de la PGE 2 .Les résultats ont été exprimés en terme de concentrations de la solution utilisée pour la provocation induisant une chute de 20 % du VEMS (CP 20 ).La PGE 2 utilisée en prétraitement a augmenté sensiblement la CP 20 LTD 4 mais pas la CP 20 méthacholine.La CP 20 LTD 4 moyenne est passée de 2,00 mg/mL (% erreur type de la moyenne 1,65) après le prétraitement avec le diluant, à 3,01 mg/mL (% erreur type de la moyenne 1,64) après le prétraitement à la PGE 2 (P=0,008).La CP 20 moyenne de la méthacholine était de 1,28 mg/mL (% erreur type de la moyenne 1,68) après le prétraitement avec le diluant et de 1,62 mg/mL (% erreur type de la moyenne 1,46) après le prétraitement à la PGE 2 (P=0,28).Ces résultats laissent croire que la PGE 2 atténue partiellement la bronchoconstriction induite par les LTD 4 ; cependant, l'ampleur de l'effet, vraisemblablement n'explique pas son atténuation de la bronchoconstriction provoquée par l'exercice et les allergènes.
Frosst) was stored at -70°C and before use was diluted in phosphate buffered saline with benzyl alcohol (pH=7.4)(Bencard) to the appropriate concentrations.The response was measured by FEV 1 performed at 30 s, 1.5 mins and 3 mins, and then every 2 mins, if necessary, until the lowest value was obtained.Once a fall in FEV 1 of 20% or greater occurred, the test was terminated and the concentration of LTD 4 required to produce a fall in FEV 1 of 20% was calculated and expressed as the LTD 4 PC 20 .After the test, two puffs of salbutamol (200 mg) were given to reverse bronchoconstriction.Analysis: Statistical analyses were performed using the STATISTICA (StatSoft Inc, Oklahoma) computer software program.Data distributions were checked for normality using Kolmogorov-Smirnoff and c 2 analysis.Because PC 20 values are log-normally distributed, log transformed methacholine and LTD 4 PC 20 s were used to compare the effect of diluent and PGE 2 .The results were also evaluated as the maximal fall in FEV 1 after the highest inhaled concentration of inhaled LTD 4 or methacholine used after diluent preteatment.FEV 1 values were not log transformed.A two-tailed paired t test was used to determine and P=0.05 was considered significant.
The results were also evaluated as the maximal fall in the FEV 1 after the highest inhaled concentration of LTD 4 and methacholine after diluent and the fall in FEV 1 after the same concentrations of the agonists after inhaled PGE 2 .According to this analysis, inhaled PGE 2 again slightly, but significantly, reduced the maximal fall in FEV 1 after inhaled LTD 4 from a mean value of 24.8% (SEM 2.8%) after diluent to 17.3% (SEM 2.8%) after PGE 2 (P=0.04)but not after inhaled methacholine, which was 27.5% (SEM 3.3%) after diluent and 22.03% (SEM 3.6%) after PGE 2 (P=0.31).The initial mean baseline FEV 1 values before diluent on the two days on which diluent was inhaled was 2.96 L (SEM 0.08), and before PGE 2 on the two days on which PGE 2 was inhaled it was 3.10 L (SEM 0.19) (P=0.43)(Figure 2).The FEV 1 significantly decreased by 0.23 L (SEM 0.08) (P=0.016) 5 mins after inhaled PGE 2 (Figure 2), but was no longer significantly reduced by 30 mins after PGE 2. Inhaled diluent had no significant effect on the FEV 1 at either 5 or 30 mins after inhalation (Figure 2).
In all subjects, inhaled PGE 2 caused transient coughing, lasting 15 to 20 s after beginning inhalation, and most subjects complained of retrosternal soreness, lasting 1 to 2 mins after beginning inhalation.

DISCUSSION
This study has demonstrated that pretreatment with inhaled PGE 2 significantly attenuates airway responsiveness to inhaled LTD 4 , but not to methacholine, in asthmatic subjects.These results suggest that inhaled PGE 2 selectively attenuates LTD 4 -induced bronchoconstrictor responses.However, the lack of effect of inhaled PGE 2 on inhaled methacholine may have resulted from the small sample size in the study.The result is, however, consistent with another study from our laboratory, which demonstrated no significant effect of inhaled PGE 2 on methacholine airway hyperresponsiveness (5).Inhaled PGE 2 caused slight, but significant, bronchoconstriction measured 5 mins after PGE 2 inhalation.This bronchoconstriction had resolved by 30 mins after the PGE 2 inhalation, immediately before the LTD 4 or methacholine inhalation.Inhaled PGE 2 has been previously shown to cause transient bronchoconstriction, even in normal subjects, lasting up to 5 mins (17).Also, as in other studies using inhaled PGE 2 in human subjects (17,18), we found that PGE 2 caused cough in all subjects, which was very transient, and retrosternal soreness in most subjects.
Previous studies have reported that inhaled PGE 2 markedly attenuates allergen-induced early responses by more than 90% and the late bronchoconstrictor response by more than 50% (7).Also, Melillo et al (5) have shown that the same dose of inhaled PGE 2 given 30 mins before exercise significantly attenuates exercise-induced bronchoconstriction by 66%.Pretreatment with LTD 4 receptor antagonists has been shown to attenuate allergen-induced asthmatic responses (19) and exercise-induced bronchoconstrictor responses (10,12) by an almost identical magnitude.Manning et al ( 4) have shown that exercise refractoriness is, at least in part, caused by LTD 4 -induced inhibitory prostaglandin release in asthmatic airways.Taken together, these studies raise the possibility that the protective effect of inhaled PGE 2 occurs through a specific effect on LTD 4 receptors, resulting in receptor antagonism.The absence of a significant effect of inhaled PGE 2 on the methacholine PC 20 is consistent with the previous findings of Melillo et al (5), and is also consistent with the fact that no tachyphylaxis to repeated challenges performed 1 h apart occurs to the cholinergic agonists methacholine (20) and acetylcholine (3) in asthmatic subjects.
Although this study has demonstrated that PGE 2 attenuates LTD 4 -induced bronchoconstriction, the magnitude of this effect shows that this mechanism is unlikely to account entirely for PGE 2 -induced attenuation of exercise and allergen-induced bronchoconstriction.If the results are analyzed as the maximal fall in FEV 1 after the highest inhaled concentration of LTD 4 , the magnitude of protection achieved by PGE 2 was small and only significant because the effect occurred in all subjects.This is in marked contrast to the major degree of protection by this dose of inhaled PGE 2 against exercise and allergen challenge.One other possible mechanism for the differences between inhaled LTD 4 and exercise or allergen challenge is that the site of action in the airway tree of inhaled LTD 4 and endogenous LTD 4 is different.This possibility cannot be discounted; however, it is unlikely to explain the lack of marked effect of inhaled PGE 2 on inhaled LTD 4 bronchoconstrictor responses, because these mediators were delivered by the same nebulizer into the same subjects.
In conclusion, our results suggest that inhaled PGE 2 partially attenuates LTD 4 -induced bronchoconstriction, an effect not seen in this or another study (5) with inhaled methacholine; however, another inhibitory effect is likely to explain its attenuation of exercise and allergen-induced bronchoconstriction.

Inhaled PGE2 and bronchoconstrictor responses Figure 1)
Can Respir J Vol 3 No 3 March/April 1996 167 Effect of inhaled prostaglandin (PG)E 2 or its diluent on leukotriene (LT)D 4 and methacholine airway responsiveness, expressed as the provocation concentration causing a 20% fall in forced expiratory volume in 1 s (PC 20 ).Pretreatment with PGE 2 caused a slight but significant increased in the LTD 4 PC 20 (P=0.008), but not the methacholine PC 20 (P=0.28) Figure 2) Effect of inhaled diluent and prostaglandin (PG)E 2 on the baseline forced expiratory volume in 1 s (FEV 1 ).PGE 2 caused a significant decrease in the FEV 1 5 mins after PGE 2 pretreatment (P=0.016), which was no longer significantly reduced 30 mins after the PGE 2 .The FEV 1 was not significantly altered after inhaled diluent at 5 mins and 30 mins