Original Paper Respiration 1992;59:72-76
Astra Draco AB, Lund, Sweden
Key Words Citric acid Cough Disodium cromoglycate Ipratropium bromide Lidocaine Terbutaline Theophylline
Selective Inhibition of Cough and Bronchoconstriction in Conscious Guinea Pigs
Abstract In guinea-pigs citric acid-induced cough and bronchoconstriction were inhib ited by p2-agonist and xanthine drugs. Lidocaine inhibited only cough. Cro moglycate and ipratropium bromide inhibited only bronchoconstriction. We conclude that cough and bronchoconstriction in guinea-pigs are distinct re flexes and that the inhibitory pharmacology of these airway reflexes may agree, in many respects, with that observed in asthmatic subjects.
Introduction
Methods
Cough and bronchoconstriction are common airway reactions frequently occurring simultaneously in airway diseases. A role for bronchial smooth muscle contraction in the genesis of cough reflexes has been suggested [1] but has not been established. In human subjects as well as in experimental animals cither cough, bronchoconstriction or both can be produced by inhaled irritants [2-4], suggest ing that these airway reflexes can be triggered independ ently. For example, a selective degeneration of capsaicinsensitive sensory neurons in guinea-pigs inhibits broncho constriction but not coughing produced by nicotine and cigarette smoke [4], Data obtained from asthmatic sub jects favour the view that cough and bronchoconstriction are distinct mechanisms: these two airway reflexes were selectively inhibited by lidocaine or cromoglycate, respec tively [5]. In the present study, we have employed conscious guinea-pigs exposed to citric acid to examine quantita tively the pharmacology of inhibition of reflex-induced cough and bronchoconstriction. Besides lidocaine we have used different anti-asthma drugs known to exert a variety of airway effects.
Received: September 16.1991 Accepted after revison: January 8,1992
Experiments on cough and bronchoconstriction on conscious gui nea-pigs were performed on Dunkin Hartley guinea-pigs of both sexes, weighing 3(X)-500 g. Unanaesthetized, unrestrained guineapigs were placed individually in a perspex chamber and exposed to nebulized aqueous solutions of citric acid (0.40 M ) for up to 7 min. The aerosols were produced by an ultrasonic nebulizer (Omron NEU 10) and had an aerodynamic mass median diameter of 0.9 pm. About 0.5 ml solution was nebulized per minute to each animal. The animals were continuously watched by a trained observer, who counted the number of coughs and noted the time to onset of bronchoconstric tion. The sounds of cough, which are clearly distinguished from sneezes, were picked up by a microphone connected to a tape-re corder, amplified and reproduced by a loudspeaker. The number of coughs produced during the first 3 min of exposure was counted. Bronchoconstriction was defined as the abrupt onset of a slow la boured breathing with exaggerated abdominal movements. We have previously shown that these signs correlate in time with a pronounced wheezing from the chest and with a significantly altered pattern of breathing recorded on a Grass polygraph [4], In preliminary experiments the optimum route of administration and pretreatment time for each drug were determined. Groups of guinea-pigs inhaled nebulized ipratropium bromide (100-300 pAf), li docaine (1-30 mM) and terbutaline (1-100 pM) for 5 min immediately before exposure to citric acid aerosol. Cromoglycate (20-60 mM) was inhaled for 10 min and the animals were allowed to rest for 20 min be fore challenge. Enprofylline (3-propyIxanthine, 4-60 umol kg ') and theophylline (30-250 pmol kg'1) were given intraperioneally (i.p.) 30
Kjell Forsberg Astra Draco A B Box 34 S—221 (M) Lund (Sweden)
© 1992 S. Karger A G . Basel 0025-7931/92/0592-0072 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/14/2017 12:48:40 PM
AL Forsberg J.-A. Karlsson C. Zackrisson C.GA. Persson
min before provocation. Control animals for the xanthines were in jected i.p. with the solvent 30 min before exposure to citric acid. In all experiments the results of the drug-treated groups were compared with their respective control group run on the same day. Data arc expressed as mean values ±SEM. Statistical differences between mean values of data obtained in groups of guinea-pigs (n = 8 in each group) were calculated by analysis of variance. If the overall F value was significant, differences between groups were evaluated by use of Student’s t test for unpaired observations. Parametric statisti cal tests could be used to evaluate cough, since we have previously shown that citric acid-induced cough in guinea-pigs is normally dis tributed 14]. A p value of less than 5% was considered statistically sig nificant. When appropriate, regression analysis was used to calculate dose ratios and ICJ0 values. The following drugs were used: acetylcholine (Sigma, USA), citric acid (Apoteksbolaget, Sweden), disodium cromoglycatc (Fisons, UK), enprofylline anhydrous (Astra Draco AB. Sweden), ipratro pium bromide (Boehringer Ingclhcim. FRG), lidocaine hydrochlo ride (Astra, Sweden), tcrbutaline sulphate (Astra Draco, Sweden), theophylline anhydrous (Astra Draco AB, Sweden). Stock solutions of drugs were made up daily in saline. All dilutions were made with 0.9% saline.
140 -
120
-
I--------------------i----------- 1------- 1 1 . 1
1
3
n -1--------------------1----------- 1--------1
10
I
1 1 1 1 I
30
100
Lidocaine, mM
Fig. 1. The effect of lidocaine aerosol (5-min exposure, immedi ately followed by provocation) on citric acid aerosol-induced cough ( • ) and bronchoconstriction (O) expressed as per cent of control value. Each dose was given to 8 guinea pigs. Note that for broncho constriction an increased inhibition is reflected as a higher percent age.
Results
240
220
-
20 0
-
180 160 1
140 -
C
8 120 -
o ^
100
-
80 60 40 -
20
-
0i--------- 1—
1— 1 1 1 1 1 1 |------------ 1—
1
3
10
1
.................. ....................1—
30
100
i
300
Terbutaline, m M
Fig.2 . The effect of terbutaline aerosol (5-min exposure, immedi ately before provocation) on citric acid aerosol-induced cough ( • ) and bronchoconstriction (O) expressed as per cent of control value. Each dose was given to 8 guinea pigs. Note that for bronchoconstric tion an increased inhibition is reflected as a higher percentage.
73
Downloaded by: Kings's College London 137.73.144.138 - 11/14/2017 12:48:40 PM
Citric acid induced significant cough and brochoconstriction. The number of coughs in twelve control groups (each comprising 8 guinea-pigs) was 5.3 ±0.4 (range 2.87.8) and the time to bronchoconstriction 123 ± 12 s (range 45-201 s). Inhaled ipratropium bromide (100 and 300 pAf) inhib ited the citric acid-induced bronchoconstriction but was without effect on cough. A concentration of 100 pAf ipra tropium bromide inhibited the bronchoconstriction caused by inhaled acetylcholine (20.4 mAf; control: 29±3 s; ipratropium bromide: 121 ± 17 s, p
Astra Draco AB, Lund, Sweden
Key Words Citric acid Cough Disodium cromoglycate Ipratropium bromide Lidocaine Terbutaline Theophylline
Selective Inhibition of Cough and Bronchoconstriction in Conscious Guinea Pigs
Abstract In guinea-pigs citric acid-induced cough and bronchoconstriction were inhib ited by p2-agonist and xanthine drugs. Lidocaine inhibited only cough. Cro moglycate and ipratropium bromide inhibited only bronchoconstriction. We conclude that cough and bronchoconstriction in guinea-pigs are distinct re flexes and that the inhibitory pharmacology of these airway reflexes may agree, in many respects, with that observed in asthmatic subjects.
Introduction
Methods
Cough and bronchoconstriction are common airway reactions frequently occurring simultaneously in airway diseases. A role for bronchial smooth muscle contraction in the genesis of cough reflexes has been suggested [1] but has not been established. In human subjects as well as in experimental animals cither cough, bronchoconstriction or both can be produced by inhaled irritants [2-4], suggest ing that these airway reflexes can be triggered independ ently. For example, a selective degeneration of capsaicinsensitive sensory neurons in guinea-pigs inhibits broncho constriction but not coughing produced by nicotine and cigarette smoke [4], Data obtained from asthmatic sub jects favour the view that cough and bronchoconstriction are distinct mechanisms: these two airway reflexes were selectively inhibited by lidocaine or cromoglycate, respec tively [5]. In the present study, we have employed conscious guinea-pigs exposed to citric acid to examine quantita tively the pharmacology of inhibition of reflex-induced cough and bronchoconstriction. Besides lidocaine we have used different anti-asthma drugs known to exert a variety of airway effects.
Received: September 16.1991 Accepted after revison: January 8,1992
Experiments on cough and bronchoconstriction on conscious gui nea-pigs were performed on Dunkin Hartley guinea-pigs of both sexes, weighing 3(X)-500 g. Unanaesthetized, unrestrained guineapigs were placed individually in a perspex chamber and exposed to nebulized aqueous solutions of citric acid (0.40 M ) for up to 7 min. The aerosols were produced by an ultrasonic nebulizer (Omron NEU 10) and had an aerodynamic mass median diameter of 0.9 pm. About 0.5 ml solution was nebulized per minute to each animal. The animals were continuously watched by a trained observer, who counted the number of coughs and noted the time to onset of bronchoconstric tion. The sounds of cough, which are clearly distinguished from sneezes, were picked up by a microphone connected to a tape-re corder, amplified and reproduced by a loudspeaker. The number of coughs produced during the first 3 min of exposure was counted. Bronchoconstriction was defined as the abrupt onset of a slow la boured breathing with exaggerated abdominal movements. We have previously shown that these signs correlate in time with a pronounced wheezing from the chest and with a significantly altered pattern of breathing recorded on a Grass polygraph [4], In preliminary experiments the optimum route of administration and pretreatment time for each drug were determined. Groups of guinea-pigs inhaled nebulized ipratropium bromide (100-300 pAf), li docaine (1-30 mM) and terbutaline (1-100 pM) for 5 min immediately before exposure to citric acid aerosol. Cromoglycate (20-60 mM) was inhaled for 10 min and the animals were allowed to rest for 20 min be fore challenge. Enprofylline (3-propyIxanthine, 4-60 umol kg ') and theophylline (30-250 pmol kg'1) were given intraperioneally (i.p.) 30
Kjell Forsberg Astra Draco A B Box 34 S—221 (M) Lund (Sweden)
© 1992 S. Karger A G . Basel 0025-7931/92/0592-0072 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/14/2017 12:48:40 PM
AL Forsberg J.-A. Karlsson C. Zackrisson C.GA. Persson
min before provocation. Control animals for the xanthines were in jected i.p. with the solvent 30 min before exposure to citric acid. In all experiments the results of the drug-treated groups were compared with their respective control group run on the same day. Data arc expressed as mean values ±SEM. Statistical differences between mean values of data obtained in groups of guinea-pigs (n = 8 in each group) were calculated by analysis of variance. If the overall F value was significant, differences between groups were evaluated by use of Student’s t test for unpaired observations. Parametric statisti cal tests could be used to evaluate cough, since we have previously shown that citric acid-induced cough in guinea-pigs is normally dis tributed 14]. A p value of less than 5% was considered statistically sig nificant. When appropriate, regression analysis was used to calculate dose ratios and ICJ0 values. The following drugs were used: acetylcholine (Sigma, USA), citric acid (Apoteksbolaget, Sweden), disodium cromoglycatc (Fisons, UK), enprofylline anhydrous (Astra Draco AB. Sweden), ipratro pium bromide (Boehringer Ingclhcim. FRG), lidocaine hydrochlo ride (Astra, Sweden), tcrbutaline sulphate (Astra Draco, Sweden), theophylline anhydrous (Astra Draco AB, Sweden). Stock solutions of drugs were made up daily in saline. All dilutions were made with 0.9% saline.
140 -
120
-
I--------------------i----------- 1------- 1 1 . 1
1
3
n -1--------------------1----------- 1--------1
10
I
1 1 1 1 I
30
100
Lidocaine, mM
Fig. 1. The effect of lidocaine aerosol (5-min exposure, immedi ately followed by provocation) on citric acid aerosol-induced cough ( • ) and bronchoconstriction (O) expressed as per cent of control value. Each dose was given to 8 guinea pigs. Note that for broncho constriction an increased inhibition is reflected as a higher percent age.
Results
240
220
-
20 0
-
180 160 1
140 -
C
8 120 -
o ^
100
-
80 60 40 -
20
-
0i--------- 1—
1— 1 1 1 1 1 1 |------------ 1—
1
3
10
1
.................. ....................1—
30
100
i
300
Terbutaline, m M
Fig.2 . The effect of terbutaline aerosol (5-min exposure, immedi ately before provocation) on citric acid aerosol-induced cough ( • ) and bronchoconstriction (O) expressed as per cent of control value. Each dose was given to 8 guinea pigs. Note that for bronchoconstric tion an increased inhibition is reflected as a higher percentage.
73
Downloaded by: Kings's College London 137.73.144.138 - 11/14/2017 12:48:40 PM
Citric acid induced significant cough and brochoconstriction. The number of coughs in twelve control groups (each comprising 8 guinea-pigs) was 5.3 ±0.4 (range 2.87.8) and the time to bronchoconstriction 123 ± 12 s (range 45-201 s). Inhaled ipratropium bromide (100 and 300 pAf) inhib ited the citric acid-induced bronchoconstriction but was without effect on cough. A concentration of 100 pAf ipra tropium bromide inhibited the bronchoconstriction caused by inhaled acetylcholine (20.4 mAf; control: 29±3 s; ipratropium bromide: 121 ± 17 s, p
