- Open Access
Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat
© Schmidt et al; licensee BioMed Central Ltd. 2008
- Received: 07 January 2008
- Accepted: 20 February 2008
- Published: 20 February 2008
Platelet activating factor and tachykinins (substance P, neurokinin A, neurokinin B) are important mediators contributing to increased airway secretion in the context of different types of respiratory diseases including acute and chronic asthma. Leukotriene receptor antagonists are recommended as add-on therapy for this disease. The cys-leukotriene-1 receptor antagonist montelukast has been used in clinical asthma therapy during the last years. Besides its inhibitory action on bronchoconstriction, only little is known about its effects on airway secretions. Therefore, the aim of this study was to evaluate the effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity.
The effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity in the rat were assessed by quantification of secreted 35SO4 labelled mucus macromolecules using the modified Ussing chamber technique.
Platelet activating factor potently stimulated airway secretion, which was completely inhibited by the platelet activating factor receptor antagonist WEB 2086 and montelukast. In contrast, montelukast had no effect on tachykinin induced tracheal secretory activity.
Cys-leukotriene-1 receptor antagonism by montelukast reverses the secretagogue properties of platelet activating factor to the same degree as the specific platelet activating factor antagonist WEB 2086 but has no influence on treacheal secretion elicited by tachykinins. These results suggest a role of montelukast in the signal transduction pathway of platelet activating factor induced secretory activity of the airways and may further explain the beneficial properties of cys-leukotriene-1 receptor antagonists.
- Platelet Activate Factor
- Mucus Secretion
- Secretagogue Effect
Increased production of airway mucus is one of the critical pathophysiological features of bronchial asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD) . Several mediators have been identified as key players in mucus hypersecretion including acetylcholine, histamine, leukotrienes, platelet activating factor (PAF), and tachykinins . The latter group belongs to a family of peptides (e.g. substance P, neurokinin A, neurokinin B) which are released from airway nerves upon stimulation . We have previously demonstrated that tachykinins are potent inducers of tracheal mucus secretion in the rat [4–6]. Furthermore, others could prove the secretagogue properties of PAF in rodents, swine, and human airway tissue [7–9]. It has been postulated that PAF has the potential to generate bioactive lipids via the 5-lipoxygenase pathway, which represents a possible mechanism mediating its secretagogue properties [10–12]. In this regard, Goswami et al. could show that PAF stimulates the secretion of respiratory glycoconjugates from human airways in culture, which was totally inhibited by the experimentally used competitive leukotriene D4 antagonist LY 171883 . The effect of clinically available cysteinyl-leukotriene-1 (cys-LT1) antagonists (montelukast, zafirlukast, or pranlukast) on PAF- or tachykinin induced secretory activity in the airways has never been evaluated. Therefore, it was the aim of this study to investigate the effects of montelukast on PAF- and tachykinin induced tracheal mucus secretion.
Pentobarbital sodium (Nembutal®) for anesthesia was obtained from Sanofi (München, Germany). Sodium azide and acetylcholine were purchased from Merck (Darmstadt, Germany). Substance P, neurokinin A, and neurokinin B were from Bachem (Heidelberg, Germany). PAF was purchased from Calbiochem (Bad Soden, Germany). WEB 2086 was from Boehringer Ingelheim (Biberach, Germany). Na2 35SO4 for radiolabelling glycoproteins was from Amersham (Braunschweig, Germany) and montelukast (MK-476) was received as a gift from Merck Frosst (Quebeck, Canada). Substance P, neurokinin A, and neurokinin B were dissolved in aqua ad injectabilia. The vehicle for PAF was ethanol. Montelukast and WEB 2086 were dissolved in dimethylsulfoxid (DMSO). Maximum concentrations of ethanol or DMSO during the experiments were 0.5%. None of the vehicles showed any significant effects on tracheal secretory activity (data not shown).
Male Sprague-Dawley rats (Harlan Winkelmann GmbH, Borchen, Germany) with an average body weight of 436 ± 42 g were used for all experiments. The experimental protocol was approved by the local animal care and use committee, and all animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals . The animals were kept in a light- and temperature controlled room and had free access to water and a rat standard diet (Altromin, Lage, Germany).
The modified Ussing chamber technique is well established for measurement of tracheal secretion and has been described in detail previously . Briefly, rats were anesthetized by an intraperitoneal injection of 70 mg*kg-1 body weight pentobarbital sodium. The trachea was excised through a ventral collar midline incision and median sternotomy and immediately transferred to an organ bath filled with medium M199 in Earle's balanced salt solution (Gibco, Eggenstein, Germany), equilibrated with carbogen gas (95% oxygen, 5% carbon dioxide). After removing the connective tissue, the trachea was opened along the paries membranaceus and mounted between the two halves of the modified Ussing chamber. According to the volume of the perfusion device, seven millilitres of medium M199 were added to the luminal (i.e. mucosal) and submucosal sides, respectively. The pH was adjusted to 7.41 and a constant temperature of 37°C was maintained during the whole experiment.
Radiolabelling and measurement of airway glycoprotein secretion
50 μCi Na2 35SO4 were added to the solution bathing at the submucosal side and allowed to equilibrate with the tissue for the duration of the experiment. After 2 h the release of bound 35SO4 to the mucosal side reaches steady state . Subsequently the luminal solution was collected every 15 minutes and replaced with fresh medium. The perfusate samples from the luminal side were collected in cellulose dialysis tubing (12,000 – 14,000 Da molecular mass cut-off, Serva, Heidelberg, Germany) and dialysed against distilled water containing unlabelled Na2SO4, to remove unincorporated 35SO4, and sodium azide (10 mg*L-1) to prevent bacterial degradation. Dialysis was complete when the radioactive count of the dialysis fluid 3 h after the last change was the same as before dialysis. The samples were transferred to plastic vials mixed with 10 ml of szintillant (Lumagel®, Baker, Deventer, Netherlands) and radioactivity was measured using a liquid szintillation counter (Rackbeta LKB 1219, LKB Instruments, Graefeling, Germany). The counts of labelled macromolecules represent the secretory activity. Former studies from our lab using high-performance liquid chromatography (HPLC) and autoradiography identified these labelled macromolecules as airway secretory glycoproteins from the submucosal glands, which were not digested by chondroitinase ABC. Thus, these macromolecules are true glycoproteins.
After two hours of incubation, samples were collected every 15 minutes. The average of two samples before pharmacological intervention represented the basal secretion rate (= 100%). Drugs were applied to the mucosal side and collections were taken 15 minutes later. Between each application, at least four samples were collected to allow the system to recover and reach a basal secretion again. In order to test the viability of the system, each experiment was finished with a stimulation of acetylcholine (1 μM), an established secretagogue for this system.
Data are expressed in percent of basal secretion ± SEM. Statistical analysis was performed with Student's t-test for paired samples. Experiments with five animals per group were performed for each experimental protocol. Data were considered significant when P < 0.05. Statistical analysis was performed using the Sigma Stat software package (Jandel Scientific, San Rafael, CA).
Effect of WEB 2086 on PAF induced tracheal secretory activity
Effect of montelukast on PAF induced tracheal secretory activity
Effect of montelukast on substance P, neurokinin A, and neurokinin B induced tracheal secretory activity
The aim of the present study was to characterize the effects of the clinically used cys-LT1 receptor antagonist montelukast on PAF- and tachykinin induced tracheal secretory activity in the rat. Our results could demonstrate that PAF potently stimulates tracheal mucus secretion. This could be completely blocked by administration of the selective PAF receptor antagonist WEB 2086 as well as montelukast. In addition, we could show that the tachykinins substance P, neurokinin A, and neurokinin B also significantly increased tracheal mucus secretion. In contrast to the inhibition of PAF induced secretion, montelukast did not modulate tachykinin stimulated secretory activity.
Recently, we demonstrated that the cys-LT1-receptor antagonist zafirlukast is a potent stimulator of tracheal secretion in the rat . In contrast, montelukast has much lower potency and does not exert secretagogue effects until concentrations of 100 μM are reached. Therefore, we used 10 μM montelukast in the present study to evaluate the effects of this cys-LT1 receptor antagonist on PAF and tachykinin stimulated tracheal secretory activity in the rat.
The naturally occurring phospholipid mediator PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is produced by a variety of inflammatory cells including neutrophils, alveolar macrophages, mast cells, eosinophils, and others. PAF originates from cleavage of membrane phospholipids by phospholipase A2 yielding lyso-PAF, which is further acetylated to form biologically active PAF. Its degradation to the inactive lyso-PAF is catalysed by a PAF-specific acetylhydrolase, which is abundantly present in plasma and intracellularly in several inflammatory cells . PAF supports the pathogenesis of many inflammatory reactions, including airway inflammation. Besides bronchoconstriction, microvascular leakage, recruitment and activation of eosinophils and airway hyperresponsiveness, PAF is seriously involved in mucus hypersecretion which is a critical feature of the inflammatory process and occurs during asthma, chronic obstructive airway disease, or pneumonia . PAF has been shown to serve as a powerful mucus secretagogue in the airways of animals and humans [13, 19]. The mechanism of PAF induced airway hypersecretion has been extensively studied during the last years. It could be demonstrated that the PAF mediated effect does not depend on a cholinergic mechanism or the generation of histamine. In contrast, accumulating evidence supports the notion that the pulmonary effects of PAF could be mediated by the secondary release of leukotrienes . It is now widely accepted that a significant amount of peptidoleukotrienes are generated in response to a PAF challenge and that these products of the arachidonic acid metabolism are at least in part responsible for the proposed PAF mediated effects [20, 21]. In addition, it could be shown that inhibition of the arachidonic acid pathway by administration of dexamethasone or inhibitors of the lipoxygenase or cyclooxygenase pathway completely blocked the secretagogue properties of PAF [7, 13, 21]. Furthermore, the experimentally used leukotriene receptor antagonist LY 171883 totally inhibited PAF-induced secretion of respiratory glycoconjugates from human airways in culture, indicating a critical role for leukotrienes in PAF induced hypersecretion . The results of the present study confirm these data and add new information concerning the clinically used cys-LT1 receptor antagonist montelukast. While the administration of montelukast alone had no effect on tracheal secretory activity, it completely inhibited PAF stimulated airway secretion in our setting. Regarding this effect, montelukast was as effective as the specific PAF receptor antagonist WEB 2086.
In addition, we could confirm earlier studies from our group indicating the secretagogue properties of the tachykinins substance P, neurokinin A, and neurokinin B in the same model. Nevertheless and unlike our previous results, neurokinin B exerted more potent secretagogue effects than neurokinin A in the present experimental series. Furthermore, mucus secretion in response to stimulation with the tachykinins was slightly lower when comparing earlier studies from our group with the results of the present investigation. It has been shown that the secretory activity of the airways could be influenced by the circadian rhythm, which could be one explanation for these differences. Moreover, the Ussing chamber position on the tracheal surface critically affects the amount of secreted mucus macromolecules and variations in that regard could also not be excluded. Crimi and colleagues have shown in human patients that montelukast abolishes the bronchoconstrictor airway response to neurokinin A, lending support to the hypothesis that tachykinins might elicit bronchoconstriction indirectly through the release of cys-LTs . In sharp contrast to the abovementioned action in the context of bronchoconstriction, montelukast did not modulate neither substance P nor neurokinin A or neurokinin B stimulated tracheal secretory activity in our setting.
In conclusion, our data show that the clinically used cys-LT1 receptor antagonist montelukast inhibits PAF induced tracheal secretory activity to the same degree as the specific PAF receptor antagonist WEB 2086. No modulating effect could be demonstrated after montelukast administration when airway secretion was stimulated by tachykinins. These findings may contribute to the beneficial effect of montelukast in the treatment of bronchial asthma.
We would thank Heike Priebe for her expert technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (Wa844/3-2).
- Danahay H, Jackson AD: Epithelial mucus-hypersecretion and respiratory disease. Curr Drug Targets Inflamm Allergy 2005, 4: 651–64. 10.2174/156801005774912851PubMedView ArticleGoogle Scholar
- Rogers DF: Physiology of airway mucus secretion and pathophysiology of hypersecretion. Respir Care 2007, 52: 1134–46. discussion 1146–9.PubMedGoogle Scholar
- Groneberg DA, Harrison S, Dinh QT, Geppetti P, Fischer A: Tachykinins in the respiratory tract. Curr Drug Targets 2006, 7: 1005–10. 10.2174/138945006778019318PubMedView ArticleGoogle Scholar
- Wagner U, Fehmann H, Bredenbroker D, Kluber D, Lange A, Wichert P: Effects of selective tachykinin-receptor antagonists on tachykinin-induced airway mucus secretion in the rat. Neuropeptides 1999, 33: 55–61. 10.1054/npep.1999.0014PubMedView ArticleGoogle Scholar
- Wagner U, Fehmann HC, Bredenbroker D, Yu F, Barth PJ, von Wichert P: Galanin and somatostatin inhibition of neurokinin A and B induced airway mucus secretion in the rat. Life Sci 1995, 57: 283–9. 10.1016/0024-3205(95)00271-7PubMedView ArticleGoogle Scholar
- Wagner U, Fehmann HC, Bredenbroker D, Yu F, Barth PJ, von Wichert P: Galanin and somatostatin inhibition of substance P-induced airway mucus secretion in the rat. Neuropeptides 1995, 28: 59–64. 10.1016/0143-4179(95)90075-6PubMedView ArticleGoogle Scholar
- Rieves RD, Goff J, Wu T, Larivee P, Logun C, Shelhamer JH: Airway epithelial cell mucin release: immunologic quantitation and response to platelet-activating factor. Am J Respir Cell Mol Biol 1992, 6: 158–67.PubMedView ArticleGoogle Scholar
- Lundgren JD, Kaliner M, Logun C, Shelhamer JH: Platelet activating factor and tracheobronchial respiratory glycoconjugate release in feline and human explants: involvement of the lipoxygenase pathway. Agents Actions 1990, 30: 329–37. 10.1007/BF01966296PubMedView ArticleGoogle Scholar
- Steiger J, Bray MA, Subramanian N: Platelet activating factor (PAF) is a potent stimulator of porcine tracheal fluid secretion in vitro. Eur J Pharmacol 1987, 142: 367–72. 10.1016/0014-2999(87)90075-6PubMedView ArticleGoogle Scholar
- Shindo K, Koide K, Fukumura M: Enhancement of leukotriene B4 release in stimulated asthmatic neutrophils by platelet activating factor. Thorax 1997, 52: 1024–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Bozza PT, Payne JL, Goulet JL, Weller PF: Mechanisms of platelet-activating factor-induced lipid body formation: requisite roles for 5-lipoxygenase and de novo protein synthesis in the compartmentalization of neutrophil lipids. J Exp Med 1996, 183: 1515–25. 10.1084/jem.183.4.1515PubMedView ArticleGoogle Scholar
- Shindo K, Koide K, Fukumura M: Platelet-activating factor increases leukotriene B4 release in stimulated alveolar macrophages from asthmatic patients. Eur Respir J 1998, 11: 1098–104. 10.1183/09031936.98.11051098PubMedView ArticleGoogle Scholar
- Goswami SK, Ohashi M, Stathas P, Marom ZM: Platelet-activating factor stimulates secretion of respiratory glycoconjugate from human airways in culture. J Allergy Clin Immunol 1989, 84: 726–34. 10.1016/0091-6749(89)90301-1PubMedView ArticleGoogle Scholar
- Science AAfLA: Guide for the Care and Use of Laboratory Animals. Bethesda, MD: National Institutes of Health; 1985.Google Scholar
- Bredenbroker D, Dyarmand D, Meingast U, Fehmann HC, Staats P, Von Wichert P, Wagner U: Effects of the nitric oxide/cGMP system compared with the cAMP system on airway mucus secretion in the rat. Eur J Pharmacol 2001, 411: 319–25. 10.1016/S0014-2999(00)00918-3PubMedView ArticleGoogle Scholar
- Schmidt R, Staats P, Groneberg DA, Wagner U: The cysteinyl-leukotriene-1 receptor antagonist zafirlukast is a potent secretagogue in rat and human airways. Eur J Pharmacol 2005, 527: 150–6. 10.1016/j.ejphar.2005.08.064PubMedView ArticleGoogle Scholar
- Chung KF: Platelet-activating factor in inflammation andpulmonary disorders. Clin Sci (Lond) 1992, 83: 127–38.Google Scholar
- Gomez FP, Rodriguez-Roisin R: Platelet-activating factor antagonists: current status in asthma. BioDrugs 2000, 14: 21–30. 10.2165/00063030-200014010-00003PubMedView ArticleGoogle Scholar
- Adler KB, Akley NJ, Glasgow WC: Platelet-activating factor provokes release of mucin-like glycoproteins from guinea pig respiratory epithelial cells via a lipoxygenase-dependent mechanism. Am J Respir Cell Mol Biol 1992, 6: 550–6.PubMedView ArticleGoogle Scholar
- Wu T, Lundgren JD, Rieves RD, Doerfler ME, Logun C, Shelhamer JH: Platelet-activating factor stimulates eicosanoid production in cultured feline tracheal explants. Exp Lung Res 1991, 17: 1079–94. 10.3109/01902149109064336PubMedView ArticleGoogle Scholar
- Adler KB, Schwarz JE, Anderson WH, Welton AF: Platelet activating factor stimulates secretion of mucin by explants of rodent airways in organ culture. Exp Lung Res 1987, 13: 25–43. 10.3109/01902148709064307PubMedView ArticleGoogle Scholar
- Crimi N, Pagano C, Palermo F, Mastruzzo C, Prosperini G, Pistorio MP, Vancheri C: Inhibitory effect of a leukotriene receptor antagonist (montelukast) on neurokinin A-induced bronchoconstriction. J Allergy Clin Immunol 2003, 111: 833–9. 10.1067/mai.2003.161PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.