Thromboxane A2 antagonist inhibits leukotriene D4-induced smooth muscle contraction in guinea-pig lung parenchyma, but not in trachea

Prostaglandins, Leukotrienes and Essential FattyAcids (1996) 55(6), 437-440 @ Pearson ProfessionalLtd 1996

Thromboxane A2 antagonist inhibits leukotriene D4-induced smooth muscle contraction in guinea.pig lung parenchyma, but not in trachea H. Aizawa, H. Inoue, K. Matsumoto, H. Koto, H. Nakano, N. Hara Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku Fukuoka 812, Japan

Summary Although the bronchoconstriction induced by leukotriene D4 (LTD4) has been reported to be partly mediated by thromboxane A2 (TXA2) in the guinea-pig airway, it is not known which part of the airway is susceptible to TXA2. In order to determine the role of TXA2 in the central and peripheral airways, we compared the effect of a TXA2 antagonist on tracheal strips to its effect on parenchymal strips of guinea-pigs. Tracheal and parenchymal strips were mounted in a 3.5 ml organ bath filled with Krebs-Henseleit solution aerated with 95% 02, 5% CO2 and kept at 37°C. After equilibration for 60 min in Krebs solution, the strip was contracted by exposure to 10-5 M of acetylcholine (ACh). Sixty minutes after ACh was eliminated, the concentration-response curve to LTD 4 (10 -9 M-10 -7 M) was obtained, and the LTD4-induced contractions were expressed as the percent of the contraction evoked by 10-5 M of ACh. We, measured the contractile response to LTD 4 in the presence or absence of the TXA~ antagonist, BAY u3405 (10 -8 M-10 -6 M). In the tracheal strips, BAY u3405 had no effect on the LTD4-induced contraction. However, in parenchymal strips, BAY u3405 significantly suppressed the contractile response to LTD4. These results suggest that in the central airway LTD 4 contracts smooth muscle directly, but that in the peripheral airway LTD4 induces smooth muscle contraction both directly and indirectly, via TXA 2. INTRODUCTION Leukotriene D 4 (LTD4) is a potent bronchoconstrictor, and

seems to play an important role in the pathogenesis of bronchial asthma.1 It has already been reported that LTD4 induces bronchoconstriction directly and indirectly, by causing the release of thromboxane A2 (TXA2) in the guinea-pig airway. 2-s Thus, LTC4, D4, and E4 administered via the pulmonary artery induced the release of cyclooxygenase products which were detected by rabbit aorta and rat stomach strips superfused by the lung effluent. This LT-induced release of TXA2 and PG-like substances was antagonized by FPL-55712. 2 The contraction of lung parenchymal strips induced by LT was inhibited by indomethacin or meclofenamic acid. 2'3 Furthermore, the bronchoconstriction induced by LT was attenuated by Received 1 May 1996 Accepted 16 May 1996 Correspondence to: H. Aizawa, MD Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812, Japan.

pretreatment with indomethacin or the TXA2 synthase inhibitor, OKY-1581 in guinea pigs in vivo. 4,~ There is, however, little documentation about the precise site in the airway that is susceptible to TXA2 in the LTD4-induced bronchoconstriction in the guinea-pig. In order to determine the role of TXA2 in the central and peripheral airways, we compared the effect of the TXA2 antagonist BAY u3405 on tracheal strips and on parenchymal strips of the guinea-pig. METHODS Protocol

Twelve guinea-pigs of either sex, weighing 500 to 800 g, were sacrificed by cervical dislocation and exsanguination. The thorax was opened and the heart, lungs, and trachea removed en bloc. Tracheal segments were opened longitudinally through the anterior aspects, and a dorsal strip was cut transversely at a length of 2-3 mm and a width of 1-1.5 ram. The mucosa and adventitial tissues were carefully removed. To measure the mecha437

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nical change, the strip was mounted in a 3.5 ml organ bath filled with Krebs-Henseleit solution aerated with 95% 02 and 5% CO2 and kept at 37°C. The composition in mM of Krebs solution was as follows: Na ÷ 131.4, K+ 5.9, Mg 2+ 1.2, Ca 2+ 2.5, el- 134.0, H2PO4- 1.2, HCO 3- 15.5, and glucose 11.5. The Krebs solution was changed every 20 min from a reservoir by a peristaltic pump (MP-3B, Eyela, Tokyo, Japan). The strip was placed vertically in the bath and its ends were tied with silk thread. One end of the strip was tied to an isometric transducer (TB-612T, Nihon Kohden Ltd, Tokyo, Japan) and the other end to a hook at the bottom of the bath. The strip was set with an initial tension of 0.5 g, which was determined in a preliminary study to be optimal for this size of strip. The subpleural parenchymal strip, 0.2 mm square and 10 m m in length, was cut from the right lower lobe and suspended using a force of 0.2 g (which was determined to be optimal in the preliminary study) in the same bath that was used for the tracheal strip. The strip was equilibrated for more than 90 min with Krebs solution, and its isometric tension was recorded continuously with a pen recorder (WT-687G, Nihon Kohden Ltd., Tokyo, Japan). After equilibration, the tracheal or parenchymal smooth muscle was contracted by exposure to 10-5 M of acetylcholine (ACh). Sixty minutes after ACh elimination, LTD4 was added to the bath, and the concentrationresponse curve to LTD4 (10-gM-10-TM) was obtained. Each concentration was given after the response to the previous concentration had reached a plateau. A control concentration-response curve was obtained from the vehicle-treated group. LTD4-induced contractions are expressed as the percent of the contraction evoked by 10-5 M of ACh. In order to determine the effects of TXA2 on LTD4induced airway smooth muscle contraction, each experiment was carried out in the presence of the TXA2 antagonist, BAY u3405, or vehicle. To avoid any effects of sympathetic nerves, each study was carried out in the presence of propranolol (10-~ M).

arithmetic mean and the standard error of mean (SEM). We used the unpaired Student's t-test for statistical analysis of the difference between the contractions induced by LTD4 in the presence and absence of BAY u3405. We considered differences to be significant when the Pvalue was less than 0.05. RESULTS Figure 1 shows an example of the original tracings showing the contraction induced by LTD4 in a guinea-pig tracheal strip. Figure 2 shows the effect of BAY u3405 on LTD4-induced smooth muscle contraction in the tracheal strips. BAY u3405 had no effects on the LTD4-induced tracheal contraction. In contrast, BAY u3405 significantly suppressed the contraction in the parenchymal strip, as shown in Figure 3. DISCUSSION In the present study, we demonstrate that a TXA2 antagonist, BAY u3405, significantly inhibits the smooth muscle contraction induced by LTD4 in the guinea-pig lung parenchyma, but not in its trachea. These results suggest that in the central airway LTD4 contracts the smooth muscle directly, but that in the peripheral airway LTD4 induces the smooth muscle contraction both directly and indirectly through TXAa generation. Piper et al reported that the generation of a TXA2-1ike substance was increased by the various LTs administered into the pulmonary artery, and that the contraction of the lung parenchymal strips induced by the LT was suppressed by indomethacin in guinea-pigs} It was also demonstrated that the bronchoconstriction induced by LT was markedly attenuated by indomethacin, and more importantly by a TXAa synthase inhibitor, OKY-1581.4,5 These observations clearly indicate that the effect of LT was partly mediated by TXA2. However, there has been 10 min

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Drugs The following drugs were used in this study: propranolol, acetylcholine hydrochloride, leukotriene D4 (Sigma, St Louis, MO) and BAY u3405 (Bayer Yakuhin Ltd, Osaka, Japan). ACh 10-5M

Statistical analysis The amplitudes of the contractions induced by L T D 4 a r e expressed as a percentage of the contraction evoked by 10-5 M ACh in each strip. The results are expressed as the

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Fig. 1 An example of an original tracing showing a contraction induced by LTD4 in the guinea-pig trachea. A tracheal strip was contracted by application of 10-2 M of acetylcholine. Sixty minutes after acetycholine washout, LTD4was added to the bath (10-9 M-10 -7 M). Each concentration was given after the response to the previous concentration had reached a plateau.

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Leukotorien D, (M) Fig. 2 The effect of BAY u3405 on LTD4-induced smooth muscle contraction in the guinea-pig trachea (n = 6). After equilibration, the tracheal strips were contracted by exposure to 10-9 M of acetylcholine. Sixty minutes after acetylcholine washout, LTD4 was added to the bath, and the concentration-response curve to LTD 4 (10 -9 M-10 -7 M) was obtained. Each concentration was given after the response to the previous concentration had reached a plateau. A control concentration-response curve was obtained from the vehicle-treated group. LTD4-induced contractions are expressed as the percent of the contraction evoked by10 5 M of acetylcholine. BAY u3405 had no effects on the LTD4-induced tracheal contraction.

Fig. 3 The effect of BAY u3405 on LTD4-induced smooth muscle contraction in guinea-pig parenchyma (n = 6). After equilibration, the parenchymal strips were contracted by exposure to 10 .5 M of acetylcholine. Sixty minutes after acetylcholine washout, LTD4 was added to the bath, and the concentration-response curve to LTD4 (10 -9 M-10 7 M) was obtained. Each concentration was given after the response to the previous concentration had reached a plateau. A control concentration-response curve was obtained from the vehicle-treated group. LTD4-induced contractions are expressed as the percent of the contraction evoked by 10-5 M of acetylcholine. BAY u3405 significantly suppressed the contraction in parenchymal strips (**P < 0.001).

little evidence indicating the site of the airways on which TXA2 mainly acts in response to LT. According to the present results, LT mainly contracts the peripheral airways by the action of TXA2. Mechanical changes of the parenchymal strips may be more similar to those of other cells instead of peripheral airway smooth muscle, because lung parenchymal strips consist of a variety of cells including airway and vascular smooth muscle, epithelial cells, endothelial cells, and connective tissues. It has been demonstrated that the responses of parenchymal strips in vitro resemble those of peripheral airways in vivo. For example, the finding that the parenchymal strips from guinea-pigs are more sensitive to histamine than ACh support interpretations made by an analysis of the differential changes in pulmonary resistance and dynamic compliance in the unanesthetized guinea-pigY TXA2 was originally described as being released from platelets, 8 but is now known to be released from other cells, including macrophages and neutrophils? In the preset study, we cannot determine which cells are responsible for the release of TXA2 in the parenchymal strips. TXA2 is known to be a potent constrictor of the airway 1° and vascular smooth muscle, 1~ and to cause platelet aggregation? 2 More importantly, TXA2 is considered to play an important role in the development of airway

hyperresponsiveness. A TXA2 synthase inhibitor has been shown to prevent the development of airway hyperresponsiveness induced by ozone, 13 LTB4,14platelet activating factor, 1~and allergens. ~ Furthermore, a TXA2 mimetic, U - 4 6 6 1 9 , has been reported to cause airway hyperresponsiveness in dogs and in humans. 13,~7Although the mechanism by which TXA2 causes airway hyperresponsiveness is not yet known, prejunctional enhancement of ACh release induced by TXAa has been reported. 18,~9 U - 4 6 6 1 9 ~8 or TXA2 released from aggregated platelets j9 causes an increase in the contractile response to vagal nerve stimulation, but no increase in the response to exogenous cholinergic agonists has been observed. The role played by TXA2 in h u m a n airway diseases is not yet known, but several studies have suggested that this agent could be an important mediator. In h u m a n subjects, increased levels of TXB2, a metabolite of TXA2, has been found in the plasma following allergen challenge 2° and in the bronchoalveolar lavage fluid following exposure to ozone. 21 Kirby et al reported that indomethacin significantly inhibits the development of bronchial hyperresponsiveness after inhalation of allergens in subjects with allergiesY BAY u3405 is a selective and potent TXA2 receptor antagonist. 23-2~ It is reported to inhibit U-46619 induced contractions in the human, guinea-pig, rat, and ferret

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a i r w a y s m o o t h m u s c l e . 24,25 BAY u 3 4 0 5 g i v e n e i t h e r intrav e n o u s l y , orally, or b y a e r o s o l h a s also b e e n r e p o r t e d to attenuate the bronchoconstriction induced by U-46619 or p r o s t a g l a n d i n D2 in g u i n e a - p i g s . 26,27 F u r t h e r m o r e , w e r e p o r t e d t h a t BAY u 3 4 0 5 a t t e n u a t e d b r o n c h i a l h y p e r r e s p o n s i v e n e s s in a p r e v i o u s study. 2s In s u m m a r y , w e h a v e d e m o n s t r a t e d t h a t BAY u 3 4 0 5 i n h i b i t s L T D 4 - i n d u c e d s m o o t h m u s c l e c o n t r a c t i o n in t h e g u i n e a - p i g l u n g p a r e n c h y m a , b u t n o t in its t r a c h e a . As LT is c o n s i d e r e d a n i m p o r t a n t m e d i a t o r in b r o n c h i a l a s t h m a , BAY u 3 4 0 5 m a y b e of v a l u e in t h e t r e a t m e n t of a s t h m a b y i n h i b i t i n g t h e effects o f LT; h o w e v e r , f u r t h e r i n v e s t i g a t i o n s are n e e d e d .

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