Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 4, 2011, pp. 445-449

Indian Journal of Pharmacology, Vol. 43, No. 4, July-August, 2011, pp. 445-449

Research Article

Effect of Montelukast on bradykinin-induced contraction of isolated tracheal smooth muscle of guinea pig

A Noor¹, MH Najmi², S Bukhtiar²

¹ Department of Pharmacology and Therapeutics, Army Medical College, National Institute of Science and Technology, Rawalpindi; Department of Pharmacology, Islamic International Medical College, Pakistan
² Department of Pharmacology and Therapeutics, Army Medical College, National Institute of Science and Technology, Rawalpindi, Pakistan

Correspondence Address: A Noor Department of Pharmacology and Therapeutics, Army Medical College, National Institute of Science and Technology, Rawalpindi; Department of Pharmacology, Islamic International Medical College Pakistan aneelanoor71@hotmail.com

Date of Submission: 31-Jul-2010
Date of Decision: 15-Apr-2011
Date of Acceptance: 25-Apr-2011

Code Number: ph11118

DOI: 10.4103/0253-7613.83119

Abstract

Keywords: ACE inhibitors, bradykinin, cough, guinea pig trachea, montelukast

Introduction

Angiotensin converting enzyme (ACE) inhibitors have been used commonly for the pharmacological treatment of hypertension, congestive cardiac failure, and diabetic nephropathy. ^[1] However, their use has been associated with respiratory adverse effects like cough, bronchoconstriction, and increased microvascular leakage. ^[2] Respiratory adverse effects associated with ACE inhibitor therapy are related to the accumulation of bradykinin and substance P in the airways, normally degraded by ACE, resulting in the enhanced sensitivity of cough reflex and reactivity of airway smooth muscle. ^[3] Bronchial C fiber stimulation by bradykinin releases a number of neuropeptides, substance P, and neurokinin A via axon reflexes, which are the chemical mediators of the cough reflex. ^[4] Another mechanism involved in the pathogenesis of ACE inhibitor-induced cough is the stimulation of Phospholipase A ₂ and the arachidonic acid (AA) pathway, by bradykinin, which results in the increased formation of prostaglandins and leukotrienes. ^[5] Bradykinin, a potent vasoactive nonapeptide and a classic mediator of inflammatory diseases like asthma, produces bronchoconstriction in asthmatic individuals as well as, when administered to experimental animals. ^[6] In spite of the well-documented bronchoconstrictor effect of bradykinin both in vivo as well as in vitro, the exact mechanism underlying this effect is still debated. It has been shown by different studies that tachykinins and AA metabolites are likely to be involved in the bronchoconstrictor effect of bradykinin. In vitro studies have shown the release of lipoxygenase metabolites from human lung fibroblasts and rat lung tissues. ^[7] Furthermore, a clinical study done on asthmatic subjects has shown an inhibitory effect of montelukast, a selective LTD ₄ antagonist, on bradykinin-induced airway hyperresponsiveness. ^[8] These observations support an interaction between bradykinin and leukotriene mediators. Keeping in view the fact that bradykinin-induced smooth muscle contraction is attributed in part to the production and release of LTD ₄ by the airway cells; the proposed study aims to explore the effect of montelukast on bradykinin-induced tracheal smooth muscle contraction in isolated guinea pig trachea. This will help find and evaluate the usefulness of montelukast in the prevention of the bradykinin-induced respiratory adverse effects encountered during ACE inhibitor therapy.

Materials and Methods

Chemicals

Chemicals used in the study include bradykinin acetate, phentolamine hydrochloride, montelukast sodium, indomethacin acetate, and propranalol hydrochloride. Solutions and dilutions of all drugs were prepared in the redistilled water, except indomethacin, which was dissolved in ethanol as it was insoluble in water.

Animals

Experiments performed were compiled with the rulings of the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council, and were approved by the PCGS committee for research, the National University of Science and Technology Islamabad Pakistan (NUST). Guinea pigs of either sex, of the Dunkin Hartley variety (500 to 600g) were housed at the animal house of the Army Medical College, Rawalpindi, NUST University, at room temperature. They were given tap water ad libitum and a standard diet for rodents. The guinea pigs were killed by cervical dislocation. The tracheal tube was cut into rings 2-3 mm wide, each containing about two cartilages. Each ring was then opened by a longitudinal cut, forming a tracheal chain with the smooth muscle in the center and the cartilaginous portion on the edges. The tissue preparation was then mounted on an isolated tissue bath of 50 ml capacity, at 37°C, containing Kreb′s Henseleit solution, with the following composition per 1000 ml: NaCl, 118.2 mM; KCl, 4.7 mM; MgSO ₄ .7H ₂ O, 1.2 mM; CaCl _2, 2.5mM; KH ₂ PO _4, 1.3mM; NaHCO _3, 25mM; Dextrose, 11.7mM. This was aerated with oxygen continuously. The tissue was allowed a period of equilibration of 45 minutes against an imposed tension of two grams. A tension of one gram was applied to the tracheal strips continuously, throughout the experiments. The trachealis muscle activity was measured with an Isometric Force Displacement transducer (Harvard model no 72-4494) and was recorded on a Four Channel Oscillograph (Harvard model no 50-9307). After the equilibration period, the tracheal muscle preparation was incubated for 15 minutes with indomethacin (10 ^-6 M), phentolamine (10 ^-5 M), and propranalol (10 ^-6 M), to eliminate the effect of the endogenous prostaglandins and catecholamines. ^[9] These drugs were added simultaneously in all the experiments and after 15 minutes, the experiments were started with this preparation.

Dose Response Curve of Bradykinin in the Absence of Montelukast

After the pre-incubation period with the baseline tension was adjusted to 1 gram, a cumulative dose response curve of bradykinin was obtained, using bradykinin of concentration 10 ^-4 M in successive doses of 11, 22, 33, 44, 55, 66, and 77 μg. When the plateau was achieved with the first dose of bradykinin, then the subsequent dose was added to the tissue bath, without washing the previous dose.

Dose Response Curve of Bradykinin in the Presence of Montelukast 0.5 μg/Ml

The cumulative dose response curve of bradykinin was obtained using the same doses of bradykinin as in the previous experiments, in the presence of montelukast sodium 0.5 μg/ml, a concentration matching to peak the plasma concentration (C _max ) achieved in adult patients, with a therapeutic dose of montelukast 10 mg orally. ^[10]

Dose Response Curve of Bradykinin in the Presence of Montelukast 1.0 μg/ml

The bradykinin dose response curve achieved with 77 μg in the presence of montelukast 0.5 μg/ml was inhibited by 38 percent; therefore, the next group of experiments was performed in the presence of higher dose of montelukast, 1 μg/ml, a concentration double the value of C _max achieved in adult patients, with a therapeutic dose of montelukast 10 mg orally. The cumulative dose response curve of bradykinin was obtained using the same doses of bradykinin as in the previous experiments. Six experiments were performed in the same manner to get six recordings in all the three groups.

Results

Observations of Individual Groups

Control group 1

Bradykinin produced a dose-dependent contraction of the guinea pig tracheal strips in the absence of montelukast pretreated with indomethacin, propranalol, and phentolamine. The effect started at the dose of 11 μg of 10 ^-4 concentration of bradykinin and reached its maximum effect at 77 μg, with a mean magnitude of response of 39 ± 6.26 mm [Table - 1]. The semi logarithm dose response curve of bradykinin was plotted with log doses on the x--axis and percent responses on the y-axis, as is shown in [Figure - 1].

Group II

In a series of six experiments performed on the tracheal tissue in the presence of 0.5 μg/ml montelukast, pretreated with indomethacin, propranalol, and phentolamine, there was no response on the first dose of bradykinin and only a minimal response -on the second dose. The mean magnitude of response at 77 μg of bradykinin was 24.17 ± 4.11 mm, respectively, causing an inhibition of 32 percent [Table - 2].The bradykinin curve in the presence of 0.5 μg/ml of montelukast, shifted to the right and downward [Figure - 2]. The deviation started with the first dose of bradykinin and continued in the entire extent of the curve.

Group III

In a series of six experiments performed on the tracheal tissues in the presence of montelukast 1 μg/ml, pretreated with indomethacin, propranalol and phentolamine, there was no response of the tracheal tissue on the first and second doses of bradykinin, and only a minimal response on the third dose of bradykinin. The mean magnitude of response for 77 g of bradykinin was 13 ± 2.6 mm [Table - 3]. The semi logarithm dose response curve of bradykinin on tracheal smooth muscle pretreated with montelukast 1 μg/ml, shifted markedly to the right and downward [Figure - 3]. The deviation started with the first dose of bradykinin and continued in the entire extent of the curve, even at the higher doses.

Comparative Observations of Groups

The mean magnitude of responses produced by each dose of bradykinin when compared between Group I and Group II, Group I and Group III, and Group II and Group III, were found to be statistically significant (P < 0.05). Percent responses calculated at each dose of bradykinin when compared between Group I and II, Group I and III, and Group II and III were found to be statistically significant (P < 0.05). Percent deviations were calculated for each dose of bradykinin used in Group I and Group II (Percent deviation A) with a mean deviation of 68.1% ± 9.8. Percent deviation calculated for each dose of bradykinin used in Group I and III (Percent deviation B) with mean deviation of 89.63% ± 5.52. Percent deviation calculated for each dose of bradykinin used in Group II and III (Percent deviation C) with mean deviation of 80.71% ± 6.65.

The difference of Percent deviations was found to be statistically significant (P < 0.05) between the groups, that is, Percent deviation A with Percent deviation B and C and Percent deviation B with Percent deviation C.

Discussion

In the present study, the dose-dependent reversible contractile effect of bradykinin on the guinea pig tracheal smooth muscle, pretreated with indomethacin, propranalol, and phentolamine started at a dose of 11 μg of bradykinin and reached its maximum at 77 μg. Dusser et al., have also reported similar effects of bradykinin on isolated ferret trachea at the concentration range of 10 ^-11 M to 10 ^-5 M. ^[11] Previous studies have shown bradykinin-induced relaxation of the guinea pig tracheal muscle with intact epithelium, and this response changed to contraction upon removal of the epithelium. ^[12] A combination of indomethacin and phosphoramidon mimics the effect of epithelium removal by blocking the effect of endogenous prostaglandins and inhibiting the neutral endopeptidase, an enzyme responsible for bradykinin metabolism. ^[13] In the present study pre-incubation of guinea pig tracheal smooth muscle, having an intact epithelium, with indomethacin, propranalol, and phentolamine mimicked the effect of epithelium removal.

Bradykinin activates nociceptive bronchopulmonary C fibers, which may mediate cough and chest tightness, the characteristic features of asthma. ^[14] Bradykinin directly activates the guinea pig tracheal afferent neurons via B ₂ receptors. ^[15]

Observations have also been made regarding the effects of bradykinin on the guinea pig tracheal strips pretreated with indomethacin, propranalol, and phentolamine in the presence of two different doses of montelukast. Results of one set of experiments in the presence of 0.5 μg/ml of montelukast, demonstrate that there is significant inhibition (38%) of bradykinin contraction and marked shifting of the bradykinin curve to the right and downwards. In the presence of 1 μg/ml of montelukast, there is 67% inhibition and a marked shifting of bradykinin dose response curve to the right and downward. The deviation started with the first dose of bradykinin and continued in the entire extent of the curve. Results of the present study reveal that leukotrienes mediate a substantial portion of bradykinin-induced tracheal smooth muscle contraction, as is evident from the inhibitory effect of montelukast. Similar results have been reported by Crimi et al., in a clinical study on asthmatics, showing a significant inhibitory effect of two tablets of 10 mg of montelukast (double the value of the therapeutic dose of montelukast in adults) against bradykinin-induced bronchoconstriction. ^[8] It was hypothesized that bradykinin-induced bronchoconstriction was either directly or indirectly mediated by the release of leukotrienes, although there was a failure to demonstrate a significant increase in urinary LTE ₄ levels, two hours after the bradykinin challenge.

Furthermore, it was hypothesized that bradykinin could up regulate the signal transduction pathway of leukotriene receptors, and this was the possible reason for the lack of increase in urinary LTE ₄ levels after the bradykinin challenge. It was the first study that had shown the significant inhibitory effect of montelukast against inhaled bradykinin. Results of the present study confirm the findings of this in vivo study, and the higher concentration used in our study in vitro, resembles the C _max , which was produced by the dose of montelukast used by Crimi et al. ^[8]

Turner et al., has shown in ventilated Hartley guinea pigs, that bradykinin-induced bronchoconstriction was significantly reduced in the presence of a neurokinin antagonist and was partially inhibited in the presence of a selective LTD ₄ receptor antagonist Pranlukast. ^[16] The results of this study are in accordance with those of the current study, with regard to the inhibitory effect of the leukotriene receptor antagonist. Likewise, Sayah et al., have reported that the bradykinin-induced contraction of the pig iris smooth muscle involves the release of prostanoids and LTD ₄ from the AA pathway. ^[17] Lindstrom et al., has shown an increased outflow of tachykinins from the sensory nerve endings on allergen-induced bradykinin stimulation. ^[18]

The illustrated antitussive effect of zafirlukast in cough-variant asthma is presumably through its anti-inflammatory action. ^[19] Bisgaard et al., reported the beneficial effect of montelukast, in the viral precipitated attack of childhood asthma exacerbation in two-to-five-year-old children. This effect of montelukast can also be related to its ability to inhibit the tachykinin-and bradykinin-induced bronchoconstriction, as viral infections trigger the bradykinin and tachykinin generation. ^[20]

The results of the present study imply the underlying mechanism in the protection afforded by montelukast against the bradykinin-induced bronchoconstriction and support the idea of an interaction between the bradykinin, tachykinin, and leukotriene mediators in the animal model of isolated guinea pig trachea. Montelukast competitively antagonized the LTD ₄ -induced contraction of the tracheal smooth muscle of guinea pig in vitro. Leukotrienes never exist preformed in the cells; rather they are formed from the breakdown of the AA attached to the phospholipids of the cell membranes. Immunological and various non-immunological stimuli including bradykinin can release AA from the membrane phospholipids by activating phopholipase A2. ^[21]

A recent study has recognized the capacity of montelukast that it directly and selectively inhibits 5-Lipoxygenase, limiting the generation of CysLT. ^[22] Keeping in view, the fact that bradykinin-induced contraction might be mediated indirectly by the release of leukotriene mediators, it can be speculated that the inhibitory effects of montelukast against the bradykinin-induced contractions might be the result of its ability to block the receptors or to cause selective inhibition of the 5-lipoxygenase enzyme. Furthermore, it is supposed that our findings about the inhibitory effect of montelukast might have a clinical relevance, considering the importance of bradykinin as a local mediator involved in a number of airway responses to different stimuli, particularly the cough and bronchospasm encountered during ACE-inhibitor therapy. In patients with sensitive airway diseases or with decreased pulmonary functions, cough has been the serious adverse reaction to ACE inhibitors. Management of ACE inhibitor-induced respiratory adverse effects is being aimed at by a number of clinical trials and experimental studies. This inhibitory effect of montelukast against bradykinin-induced contraction may open a new area of research with regard to the management of bradykinin-induced respiratory adverse effects encountered during ACE inhibitor therapy. As leukotriene receptor antagonists have been seen to be useful in the management of cough in patients with cough-variant asthma, the effective role of montelukast in the management of bradykinin-induced cough with ACE inhibitor therapy is suggested.

In conclusion, the significant inhibitory effect of montelukast against bradykinin-induced tracheal smooth muscle contraction has been explored in the present study and supports an interaction between bradykinin and leukotriene mediators. Further studies should explore and elucidate the exact mechanism underlying this inhibitory effect of montelukast. Simultaneous multicenter clinical trials in human subjects facing respiratory adverse effects during ACE inhibitor therapy could also be an option to find the role of leukotriene receptor antagonists in the prevention of these adverse effects.

Acknowledgment

We are grateful to Mr. Tauseef Ahmed for the technical assistance he provided throughout the research study and to Dr. Abdul Hannan for his assistance in conducting the statistical analyses of the present study.

NUST University Pakistan has provided financial support for this research study.

References

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