Copyright© ERS Journals Ltd 1993 Eur Respir J, 1993, 6, 434-439 European Respiratory Journal Printed in UK - all rights reserved ISSN 0903 - 1936 Effect of frusemide on bradykinin- and capsaicin-induced contraction of the guinea-pig trachea M. Molimard, C. Advenier Effect of frusemide on bradykinin- and capsaicin-induced contraction of the guinea-pig Laboratoire de Pharmacologie, Institut trachea. M. Molimard, C. Advenier. ©ERS Journals Ltd 1993. Biomedical des Cordeliers, Paris, France. ABSTRACT: Frusemide, a loop diuretic, inhibits the bronchial response to vari­ ous bronchoconstrictor stimuli in asthmatic subjects. The underlying mechanisms Correspondence: M. Molimard Laboratoire de Pharrnacologie remain unclear. Institut Biomedical des Cordeliers In order to determine whether frusemide inhibits pharmacologically induced 15, rue de l'Ecole de Medecine C-tibre stimulation, we investigated the effect of frusemide on bradykinin-, capsai­ F-75270 Paris Cedex 06 cin-, neurokinin A-, and substance P-induced contraction of the guinea-pig isolated France. trachea. Frusemide 10·5 and 10·4 M produced a significant inhibition of concentration­ Keywords: Airways smooth muscle response curves to bradykinin, which was markedly reduced by indomethacin 10"" asthma M. Frusemide significantly reduced capsaicin-induced contraction only in the bradykinin presence of indomethacin 10·6 M. Neurokinin A- and substance P-induced contrac­ capsaicin frusemide tions were not affected by frusemide and/or indomethacin. prostaglandin Our data suggest that a cyclo-oxygenase pathway is involved in the inhibition by frusemide of the bradykinin-induced contraction, but not in the inhibition of the Received: December 10 1991 capsaicin-induced contraction. accepted after revision December 9 1992. Eur Respir J., 1993, 6, 434-439. Inhalation of the loop diuretic frusemide has been Material and methods shown to inhibit bronchoconstrictor response to exercise [1], ultrasonically nebulized distilled water [2], antigen [3- 5], metabisulphite [6, 7] or adenosine [8]. The mecha­ Guinea-pig isolated trachea nism of this action remains uncertain. Frusemide is unlikely to inhibit smooth muscle contraction directly, Male guinea-pigs weighing 250-350 g were killed by since it does not influence the bronchoconstriction induced cervical dislocation. The trachea was removed and cut by histamine, KC!, and hypertonic NaCI in vitro [9]. The into rings. In some experiments, the epithelium was re­ effect of frusemide could be due to inhibition of the moved by gently rubbing the luminal surface with a cot­ Na+/CI- cotransport process, as suggested by BIANco et al. ton-tipped applicator soaked in Kreb's solution, according [ 1]. Frusemide might also act by releasing broncho­ to the method described by DEVILLIER et al. [18]. The di!atator prostaglandins from airway epithelium, as has preparations were mounted isometrically and subjected to been shown on vascular endothelium and kidney tubular an initial tension of 1.50 g, in a Kreb's solution at 37°C epithelium [10, 11]. Recently, frusemide has been shown bubbled with a 95% 0 2 and 5% C02 mixture. Kreb's so­ to inhibit, in the presence of indomethacin, the contrac­ lution was composed of (mM): NaCI 113; KC! 4.7; CaC12 tion induced by electrical field stimulation of guinea-pig 1.9; MgS04 1.2; KH2P04 1.2; NaHC03 25; glucose 11.5. bronchial smooth muscle [12]. Following equilibration for 1.25 h, the resting tension was Afferent vagal C-fibres are stimulated by bradykinin between 0.8-1.5 g. [13, 14], and capsaicin [15], and induce the release of Tensions were measured with Celaster UF-1 gauges multiple tachykinins, such as substance P (SP) and neu­ connected to Celaster AC-261 amplifiers. Analogical and rokinin A (NKA) [14-17]. The aim of the present study digital signal acquisition, enabling treatment of pharma­ was to investigate the possible inhibitory effect of fruse­ cological data, was performed using the Moise 3 program mide on neurally evoked bronchoconstrictor pathways. (Dei Lierre, 77290 Mitry-Mory, France). We studied the effect of frusemide on bradykinin-, cap­ Tracheal rings were first contracted to maximal ten­ saicin-, SP- and NKA-induced bronchoconstriction in sion with acetylcholine 1 mM, then relaxed with theo­ guinea-pig. phylline 3 mM to sensitize and stabilize the preparations. FRUSEMIDE AND GUINEA-PIG TRACHEA 435 Experiments were separated by intervals of a least 1 h. a Experiments were conducted on parallel groups of 4-8 50 rings, one ring serving as control. Each of the other rings was incubated for I h with a given concentration of the reagents or substances tested (frusemide (L0·5 and 104 M) * and/or indomethacin (I Q-6 M)). Following this incubation, ::2 c:: E and in the absence (control) or presence of reagents or .Q ~ t:>..r:: substances tested, a range of one of the following con­ rou 25 10 6 -E< traction-inducing agents (bradykinin (I0- -10· M), cap­ 0 saicin (10-9-I0-6 M), SP (10·9-10·6 M), NKA u g? ~ (10·9-10·6 M)) was established. Thereafter, acetylcholine 0 l mM was added to the bath to obtain 100% maximal contraction. The effects of the contractile agents were expressed as 0 a percentage of the effects of acetylcholine I mM. -10 -9 -8 -7 -6 -5 Bradykinin log M b Statistical analysis of results 50 All values in the text and figures are mean±sEM. Sta­ tistical analysis was performed using variance analysis and Student's t-test for paired or unpaired data. ::2 c:: E .Q ,....- 't)..r::rou Drugs and chemicals -E<( 25 t 8g? ~ The substances used were: frusemide (Hoechst), ace­ 0 t tylcholine (Pharmacie centrale des hopitaux, Paris, France); indomethacin (Merck); substance P, neurokinin A, bradykinin, capsaicin (Sigma, St. Louis, USA); theo­ 0 +-~~~--rL~.-~-.--~.-~~ phylline sodium anisate (Bruneau, Paris, France). All -11 -10 -9 -8 -7 -6 -5 drugs were dissolved in distilled water and then diluted in saline for the in vivo studies, or in Kreb's solution for Bradykinin log M the in vitro studies, except for indomethacin which was c dissolved in ethanol, and then diluted in such a way that 50 the amounts of ethanol did not alter acetylcholine reac­ tivity. Results Effect of frusemide on concentration-response curves to bradykinin on the guinea-pig trachea Bradykinin induced a transient contraction within 5 min after injection, followed by a relaxation. We studied the 0 +-~--T-~--.-~-.--~-.--.-~ cumulative contractile effect of bradykinin by adding the -10 -9 -8 -7 -6 -5 next concentration of bradykinin when the induced con­ Bradykinin log M traction reached its maximum. Figure I a shows that epithelium removal significantly Fig. I. - a) Concentration response-curves to bradykinin on enhanced the concentration response curve to bradykinin intact (.A.) and denuded (0 guinea-pig isolated trachea. on guinea-pig trachea. On epithelium-denuded trachea b) Influence of frusemide (lO·' ( .) and 10·• (6) M) on bradykinin 5 4 concentration-response curves on guinea-pig isolated trachea (fig. Ib and c), frusemide IQ- and 10 M induced a sig­ without epithelium. 0: control. c) Influence of indomethacin to·• nificant decrease of bradykinin (BK) concentration­ M on bradykinin concentration-response curves on guinea-pig de­ response curves (fig. Ib); indomethacin IQ-6 M did not nuded trachea pretreated by frusemide IO·' M (.6.).0: control (no significantly influence bradykinin control concentration­ treatment); 6: frusemide 10·• M alone; 0: indomethacin 10·6 M response curves, but significantly inhibited the decrease alone. Values are mean±sEM (n=6). Significant differences with control are indicated by: *: p<0.05; * : p<0.02; t: p<O.Ol except of bradykinin concentration-response curves induced by for figure c where the comparison was made with indomethacin frusemide (fig. le) 10·6 M. ACh: acetylcholine. 436 M. MOLIMARD, C. ADVENIER a b 100 100 * :::2: 75 :::2: 75 c::: E c::: E o~ o~ U...c u~ -E<<U() 50 -E<<U() 50 8~ 8~ ~0 ~0 25 25 0 0 -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 Capsaicin log M Capsaicin log M c d 100 100 75 :::2: 75 :::2: c::: c::: E E o~ o~ ·u ..c ·u ...c cuo Ec:t:<U() -E<t: 50 50 8~ 8~ ~0 ~0 25 - 25 0 o~~--~~--,-~r-,---~~ -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 Capsaicin log M Capsaicin log M Fig. 2. - Effect of frusemide (I0-3 M) on capsaicin concentration-response curves without (a, c, e) or with indomethacin 10-• M pre­ treatment (b, d, 0) on intact (a, b) and denuded (c, d) guinea-pig isolated trachea. .1.: indomethacin I0-6 M alone. 0: control (no treat­ ment). Values are mean±sEM (n=6). Significant differences with capsaicin response curves in the presence of indomethacin (.1.) indicated by *: p<0.05. a b 100 100 75 75 c:::::::i: c:::::::i: 0 c::: 0 c::: u~ ·u~ ~0 50 ~0 50 §<t: §<t: ()~ ()~ 25 25 0 0 -10 -9 -8 -7 -6 -10 -9 -8 -7 -6 Substance P log M Neurokinin A log M Fig. 3. - a) Influence of frusemide 10-< M (A) on substance-P concentration-response curves on guinea-pig isolated trachea without epithelium pretreated by indomethacin. b) Influence of frusemide 10-' M (A) on neurokinin A concentration-response curves on guinea­ pig denuded isolated trachea pretreated by indomethacin_ Values are mean±sEM (n=6 and n=5, respectively). There was no significant difference with substance-P or neurokinin A control response curves (Q). FRUSEMIDE AND GUINEA-PIG TRACHEA 437 Effect of frusemide on concentration-response curves to important in the secondary relaxation induced by brady­ capsaicin on the guinea-pig trachea kinin on intact guinea-pig trachea, and considered PGI2 to be less important in the response to bradykinin. This Administration of capsaicin (1 o-s to I0-5 M) produced a is in agreement with MIZRAHI et al.