Pulmonary Pharmacology (1990) 3 161-166 (01990 Longman Group UK Ltd
0952-0600/90/0003-0161/$10 .00
PULMONARY PHARMACOLOGY
Airway Hyperreactivity Induced by Active Cigarette Smoke Exposure in Guinea-pigs : Possible Role of Sensory Neuropeptides L . Daffonchio, A . Hernandez, L . Gallico, C . Omini Institutes of Pharmacological Sciences and Biological Sciences, Universities of Milan and Pisa, Italy
SUMMARY. Exposure to cigarette smoke is associated with increased airway responsiveness to different stimuli, both in human and animal studies . However, the mechanisms involved in the pathogenesis of smoke-induced airway hyperreactivity are less clear. We investigated the development of airway hyperreactivity induced by active cigarette smoke exposure in anaesthetised guinea-pigs and the possible mechanisms involved . Active inhalation of cigarette smoke (15 s/min for 10 min) potentiated the bronchocontractile effect of acetylcholine (Ach), indicating the occurrence of airway hyperreactivity . This phenomenon appeared within 5 min and lasted up to 50 min after smoke exposure . Smoke induced airway hyperreactivity was a non-specific phenomenon, involving an enhanced responsiveness to both Ach and histamine (Hist) . Recruitment of proinflammatory cells into the airway lumen, as revealed by the analysis of bronchoalveolar lavage fluid, paralleled the development of the hyperreactive phenomenon, suggesting a relationship between the inflammatory reaction and the genesis of smoke-induced airway hyperreactivity . Cervical bilateral vagotomy did not modify either the degree and the time-course of smoke induced airway hyperreactivity. Moreover, atropine treatment did not affect the increase in Hist response due to smoke inhalation . On the other hand, depletion of substance P due to capsaicin pretreatment almost completely prevented the capacity of cigarette smoke to potentiate Ach induced bronchoconstriction . Cyclo-oxygenase inhibition, by indomethacin pretreatment, reduced the time course of the hyperreactivity induced by smoke inhalation . Our results clearly demonstrate the occurrence of airway hyperreactivity triggered by active cigarette smoke exposure . Moreover, the data obtained suggest a predominant role for substance P and related peptides in the pathogenesis of smoke induced increase in airway responsiveness .
INTRODUCTION
this phenomenon after exposure to cigarette smoke in animals and the possible mechanisms involved are less clear .' 1,12 Therefore, we verified the capacity of cigarette smoke to induce airway hyperreactivity using a model of active smoke exposure in anaesthetised guineapigs . Moreover, we characterised this phenomenon and its possible link with other pathological modifications within the lung such as the recruitment of inflammatory cells . An inflammatory reaction localised to the lung has been in fact associated with the development of airway hyperreactivity in several conditions .' 3 Among different inflammatory mediators, eicosanoids have been claimed to play a role in the genesis of airway hyperreactivity ." Therefore, we investigated the involvement of prostaglandins in airway hyperreactivity induced by cigarette smoke in our model . Other experiments were carried out in vagotomised, atropine treated or capsaicin desensitised guinea-pigs in order to verify the possible role of vagal or substance P-mediated local reflexes in the genesis of the hyperreactive phenomenon . Cigarette smoke has been in fact demonstrated to induce local mucosal reactions and bronchial smooth muscle spasm via the activation of sensory reflexes .' 5
Airway hyperreactivity is a characteristic feature of asthma, shown by a non-specific enhancement of airway responsiveness to different pharmacological and environmental stimuli .' This pathological event has been commonly associated with the anaphylactic reaction triggered in both experimental animals and men . 2,3 However, airway hyperreactivity has been shown to develop also after exposure to several air pollutants .' ,' , ' Cigarette smoke is a well recognised pollutant and the health consequences of active cigarette smoking are documented by extensive experimental and epidemiological data .' . Within the respiratory system, pathological modifications such as increased airway resistance, smooth muscle hypertrophy, goblet cell hyperplasia and inflammation have been demonstrated after smoke exposure .' Increased airway responsiveness to different stimuli has also been reported in smokers; 8-10 however, the occurrence of Address correspondence to Luisa Daffonchio, Institute of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy . 161
1 62
Pulmonary Pharmacology
MATERIALS AND METHODS Active smoke exposure Male Dunkin-Hartley guinea-pigs (450-550 g; Rodentia, Italy), were randomly assigned to the different experimental groups. Animals were anaesthetised with urethane (1 .2 g/Kg i.p .), tracheostomised and ventilated at 50 strokes/min with 1 ml of air per 100 g body weight (Rodent Ventilator, Basile, Italy) . A jugular vein was also cannulated for drug administration . In order to suppress the spontaneous breathing, animals received pancuronium bromide (2 mg/Kg i.v .) . Pulmonary inflation pressure (PIP, mmHg), an index of intrathoracic airway calibre, was measured through a pressure transducer (Bentley Trantec 800) connected to the inspiration line and signals displayed on a Basile 7051 pen recorder . The smoke was generated by high tar commercially available cigarette (Stop, Monopoli di Stato, Italy) . A cigarette was directly connected to the ventilation circuit and the smoke delivered to the animals for 15 s/min for 10 min . The amount of smoke was monitored through measurements of one of its representative components, i .e . carbon monoxide (CO) . CO % volume was measured with a CO detector tube (Auergesellschaft GMBH, Berlin) . The average CO % volume delivered during smoke exposure was 0 .03 ± 0 .01 % at the beginning and 0 .73+0 .03% at the end of the cigarette (n = 3) . Functional studies The development of airway hyperreactivity was assessed as the increase in the bronchoconstriction induced by acetylcholine (Ach) after active smoke exposure . A single dose of Ach, ranging between 10 and 30 µg/Kg i .v ., was chosen at the beginning of the experiment in order to obtain a comparable bronchoconstriction in each animal (about 7-8 mmHg) . The selected dose was then administered at 15 min interval ; when at least three constant responses were obtained, animals were exposed to cigarette smoke and the bronchocontractile effect of Ach tested again at 5, 20, 35 and 50 min after smoke exposure . Preliminary experiments revealed that Ach induced bronchoconstriction was reproducible for at least 10 responses . As an example, three subsequent responses registered at 15 min interval were 7 .31 ±0 .95, 8.61 ± 0 .91 and 8 .44 ± 0 .91 mmHg (n = 16) . In some experiments, dose-response curves for Ach (3-300 µg/Kg i .v .) and histamine (Hist, 1-100 gg/Kg i.v .) were constructed subsequently 5 min after smoke exposure and at the same time interval after initial stabilisation in control animals . The bronchoconstriction was calculated at the peak effect and expressed as the increase in PIP over the basal value .
Bronchoolveolar lavage Bronchoalveolar lavage (BAL) was performed in separate animals 5 and 50 min after active smoke exposure and at 50 min after initial stabilization in controls . BAL was performed by gently washing the lung through the trachea with 10 ml x 3 times of saline (37°C) . Fluid recovery was greater than 97% in all the samples . BAL fluid was centrifuged at 800 g for 10 min and the pellet resuspended in phosphate buffered saline . Total cell count was determined after dilution with Trypan-blue (Merck, 0 .5% in 0 .2% physiological solution) by mean of `improved Neubauer' counting chamber and phase contrast microscopy . Differential cell count was evaluated on the same samples stained with May-Grunwald-Losung (Merck) and GiemsaLosung (Merck) under light microscopy, counting 500 cells in each sample .
Pharmacological treatments Vagotomy was obtained cutting both cervical vagi at the beginning of the experiment . Capsaicin desensitisation was performed according to the modified method of Gamse et al ." Increasing doses of capsaicin (0 .3, 0.6, 1 .2 and 2 .4 mg/Kg in the first day ; 2 .5, 10, 15 and 20 mg/kg during the second day) were injected s .c . at 2 h interval . 15 min before each capsaicin injection, animals were treated i .p . with a cocktail of atropine (1 .25 mg/Kg), pyrilamine (2 .5 mg/Kg), aminophylline (10 mg/Kg) and salbutamol (0 .1 mg/Kg). In order to minimise the irritation symptoms due to capsaicin, pentobarbitone sodium was injected 15 min before the first five injections at the dose of 10-20 mg/Kg i .p . 80% of the animals survived this treatment and were used 6-10 days later . The effectiveness of the desensitisation was verified administering capsaicin (10 µg/Kg i .v .) at the end of the experiment . This dose produced a sustained bronchoconstriction in normal animals (mean increase in PIP 30.29±2 .18 mmHg; n=5) while it did not affect PIP in any of the desensitised guinea-pigs . Indomethacin (5 mg/Kg i.v .) was administered I h before smoke exposure . Atropine was injected 15 min before smoke exposure at the dose of 2 mg/Kg i .v . Statistical analysis Data were expressed as mean ± SEM from (n) replications . Dose-response curves were compared by mean of a computer aided program according to the variance analysis for regression lines ." This procedure allows to calculate the dose-ratio and its 95% confidence limits . Other comparisons were performed either with paired Student's t-test and one way Dunnett's test . A probability level of p
0952-0600/90/0003-0161/$10 .00
PULMONARY PHARMACOLOGY
Airway Hyperreactivity Induced by Active Cigarette Smoke Exposure in Guinea-pigs : Possible Role of Sensory Neuropeptides L . Daffonchio, A . Hernandez, L . Gallico, C . Omini Institutes of Pharmacological Sciences and Biological Sciences, Universities of Milan and Pisa, Italy
SUMMARY. Exposure to cigarette smoke is associated with increased airway responsiveness to different stimuli, both in human and animal studies . However, the mechanisms involved in the pathogenesis of smoke-induced airway hyperreactivity are less clear. We investigated the development of airway hyperreactivity induced by active cigarette smoke exposure in anaesthetised guinea-pigs and the possible mechanisms involved . Active inhalation of cigarette smoke (15 s/min for 10 min) potentiated the bronchocontractile effect of acetylcholine (Ach), indicating the occurrence of airway hyperreactivity . This phenomenon appeared within 5 min and lasted up to 50 min after smoke exposure . Smoke induced airway hyperreactivity was a non-specific phenomenon, involving an enhanced responsiveness to both Ach and histamine (Hist) . Recruitment of proinflammatory cells into the airway lumen, as revealed by the analysis of bronchoalveolar lavage fluid, paralleled the development of the hyperreactive phenomenon, suggesting a relationship between the inflammatory reaction and the genesis of smoke-induced airway hyperreactivity . Cervical bilateral vagotomy did not modify either the degree and the time-course of smoke induced airway hyperreactivity. Moreover, atropine treatment did not affect the increase in Hist response due to smoke inhalation . On the other hand, depletion of substance P due to capsaicin pretreatment almost completely prevented the capacity of cigarette smoke to potentiate Ach induced bronchoconstriction . Cyclo-oxygenase inhibition, by indomethacin pretreatment, reduced the time course of the hyperreactivity induced by smoke inhalation . Our results clearly demonstrate the occurrence of airway hyperreactivity triggered by active cigarette smoke exposure . Moreover, the data obtained suggest a predominant role for substance P and related peptides in the pathogenesis of smoke induced increase in airway responsiveness .
INTRODUCTION
this phenomenon after exposure to cigarette smoke in animals and the possible mechanisms involved are less clear .' 1,12 Therefore, we verified the capacity of cigarette smoke to induce airway hyperreactivity using a model of active smoke exposure in anaesthetised guineapigs . Moreover, we characterised this phenomenon and its possible link with other pathological modifications within the lung such as the recruitment of inflammatory cells . An inflammatory reaction localised to the lung has been in fact associated with the development of airway hyperreactivity in several conditions .' 3 Among different inflammatory mediators, eicosanoids have been claimed to play a role in the genesis of airway hyperreactivity ." Therefore, we investigated the involvement of prostaglandins in airway hyperreactivity induced by cigarette smoke in our model . Other experiments were carried out in vagotomised, atropine treated or capsaicin desensitised guinea-pigs in order to verify the possible role of vagal or substance P-mediated local reflexes in the genesis of the hyperreactive phenomenon . Cigarette smoke has been in fact demonstrated to induce local mucosal reactions and bronchial smooth muscle spasm via the activation of sensory reflexes .' 5
Airway hyperreactivity is a characteristic feature of asthma, shown by a non-specific enhancement of airway responsiveness to different pharmacological and environmental stimuli .' This pathological event has been commonly associated with the anaphylactic reaction triggered in both experimental animals and men . 2,3 However, airway hyperreactivity has been shown to develop also after exposure to several air pollutants .' ,' , ' Cigarette smoke is a well recognised pollutant and the health consequences of active cigarette smoking are documented by extensive experimental and epidemiological data .' . Within the respiratory system, pathological modifications such as increased airway resistance, smooth muscle hypertrophy, goblet cell hyperplasia and inflammation have been demonstrated after smoke exposure .' Increased airway responsiveness to different stimuli has also been reported in smokers; 8-10 however, the occurrence of Address correspondence to Luisa Daffonchio, Institute of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy . 161
1 62
Pulmonary Pharmacology
MATERIALS AND METHODS Active smoke exposure Male Dunkin-Hartley guinea-pigs (450-550 g; Rodentia, Italy), were randomly assigned to the different experimental groups. Animals were anaesthetised with urethane (1 .2 g/Kg i.p .), tracheostomised and ventilated at 50 strokes/min with 1 ml of air per 100 g body weight (Rodent Ventilator, Basile, Italy) . A jugular vein was also cannulated for drug administration . In order to suppress the spontaneous breathing, animals received pancuronium bromide (2 mg/Kg i.v .) . Pulmonary inflation pressure (PIP, mmHg), an index of intrathoracic airway calibre, was measured through a pressure transducer (Bentley Trantec 800) connected to the inspiration line and signals displayed on a Basile 7051 pen recorder . The smoke was generated by high tar commercially available cigarette (Stop, Monopoli di Stato, Italy) . A cigarette was directly connected to the ventilation circuit and the smoke delivered to the animals for 15 s/min for 10 min . The amount of smoke was monitored through measurements of one of its representative components, i .e . carbon monoxide (CO) . CO % volume was measured with a CO detector tube (Auergesellschaft GMBH, Berlin) . The average CO % volume delivered during smoke exposure was 0 .03 ± 0 .01 % at the beginning and 0 .73+0 .03% at the end of the cigarette (n = 3) . Functional studies The development of airway hyperreactivity was assessed as the increase in the bronchoconstriction induced by acetylcholine (Ach) after active smoke exposure . A single dose of Ach, ranging between 10 and 30 µg/Kg i .v ., was chosen at the beginning of the experiment in order to obtain a comparable bronchoconstriction in each animal (about 7-8 mmHg) . The selected dose was then administered at 15 min interval ; when at least three constant responses were obtained, animals were exposed to cigarette smoke and the bronchocontractile effect of Ach tested again at 5, 20, 35 and 50 min after smoke exposure . Preliminary experiments revealed that Ach induced bronchoconstriction was reproducible for at least 10 responses . As an example, three subsequent responses registered at 15 min interval were 7 .31 ±0 .95, 8.61 ± 0 .91 and 8 .44 ± 0 .91 mmHg (n = 16) . In some experiments, dose-response curves for Ach (3-300 µg/Kg i .v .) and histamine (Hist, 1-100 gg/Kg i.v .) were constructed subsequently 5 min after smoke exposure and at the same time interval after initial stabilisation in control animals . The bronchoconstriction was calculated at the peak effect and expressed as the increase in PIP over the basal value .
Bronchoolveolar lavage Bronchoalveolar lavage (BAL) was performed in separate animals 5 and 50 min after active smoke exposure and at 50 min after initial stabilization in controls . BAL was performed by gently washing the lung through the trachea with 10 ml x 3 times of saline (37°C) . Fluid recovery was greater than 97% in all the samples . BAL fluid was centrifuged at 800 g for 10 min and the pellet resuspended in phosphate buffered saline . Total cell count was determined after dilution with Trypan-blue (Merck, 0 .5% in 0 .2% physiological solution) by mean of `improved Neubauer' counting chamber and phase contrast microscopy . Differential cell count was evaluated on the same samples stained with May-Grunwald-Losung (Merck) and GiemsaLosung (Merck) under light microscopy, counting 500 cells in each sample .
Pharmacological treatments Vagotomy was obtained cutting both cervical vagi at the beginning of the experiment . Capsaicin desensitisation was performed according to the modified method of Gamse et al ." Increasing doses of capsaicin (0 .3, 0.6, 1 .2 and 2 .4 mg/Kg in the first day ; 2 .5, 10, 15 and 20 mg/kg during the second day) were injected s .c . at 2 h interval . 15 min before each capsaicin injection, animals were treated i .p . with a cocktail of atropine (1 .25 mg/Kg), pyrilamine (2 .5 mg/Kg), aminophylline (10 mg/Kg) and salbutamol (0 .1 mg/Kg). In order to minimise the irritation symptoms due to capsaicin, pentobarbitone sodium was injected 15 min before the first five injections at the dose of 10-20 mg/Kg i .p . 80% of the animals survived this treatment and were used 6-10 days later . The effectiveness of the desensitisation was verified administering capsaicin (10 µg/Kg i .v .) at the end of the experiment . This dose produced a sustained bronchoconstriction in normal animals (mean increase in PIP 30.29±2 .18 mmHg; n=5) while it did not affect PIP in any of the desensitised guinea-pigs . Indomethacin (5 mg/Kg i.v .) was administered I h before smoke exposure . Atropine was injected 15 min before smoke exposure at the dose of 2 mg/Kg i .v . Statistical analysis Data were expressed as mean ± SEM from (n) replications . Dose-response curves were compared by mean of a computer aided program according to the variance analysis for regression lines ." This procedure allows to calculate the dose-ratio and its 95% confidence limits . Other comparisons were performed either with paired Student's t-test and one way Dunnett's test . A probability level of p
