Br. J. Pharmacol. (1992), 105, 448-452
C.) Macmillan Press Ltd, 1992
Capsaicin-induced bronchoconstriction in the guinea-pig: contribution of vagal cholinergic reflexes, local axon reflexes and their modulation by BW443C81 P. Buchan & J.J. Adcock Department of Pharmacology, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS 1 The objective of the study was to investigate the central vagal and local axon reflex components of bronchoconstrictor responses evoked by inhalation of capsaicin aerosol in anaesthetized guinea-pigs. This was accomplished by comparing the effects of bilateral vagotomy, atropine and the peripherally-acting polar enkephalin analogue, BW443C81, on bronchoconstrictor responses evoked by capsaicin. The effects of codeine were also determined. 2 Aerosols of capsaicin were generated from a 0.9 pg ml-' solution. Inhalation of capsaicin aerosol in 5, 10 and 15 breaths evoked dose-related bronchoconstrictor responses. The responses were immediate in onset and of extended duration. 3 Capsaicin-induced bronchoconstrictor responses were significantly inhibited following bilateral vagotomy or atropine (0.3 mgkg-1, i.v.) pretreatment by 46% + 14% (P < 0.05) and 59% + 13% (P < 0.01), respectively. 4 Administration of BW443C81 by intravenous infusion (3, 30 and lOOpgkg- min-1) caused a significant inhibition of capsaicin-induced bronchoconstrictor responses which achieved a greater maximum than either bilateral vagotomy or atropine. Codeine (l00 pgkg-'min-t, i.v.) did not significantly inhibit the bronchoconstrictor responses. 5 Inhibition of capsaicin-induced bronchoconstrictor responses by BW443C81 (30pgkg-1min-1, i.v.) was significantly (P < 0.05) reduced by the peripherally-acting opioid antagonist N-methyl nalorphine (l00pgkg- min- i.v.). 6 These results show that capsaicin-induced bronchoconstrictor responses are mediated by at least two mechanisms, a vagal and/or cholinergic reflex pathway and a non-cholinergic pathway. BW443C81, but not codeine, significantly inhibited (P < 0.005) both mechanisms of capsaicin-induced bronchoconstriction probably by an action on peripheral opioid receptors located on vagal sensory nerves. Keywords: Reflex bronchoconstriction; inhalation; capsaicin; sensory nerves ,
Introduction Stimulation of C-fibre afferents in the lung may contribute to a variety of airway reflexes including cough (Winning et al., 1986; Karlsson et al., 1988; Fuller et al., 1988), bronchoconstriction (Forsberg & Karlsson, 1986), mucus secretion (Davis et al., 1982) as well as changes in breathing patterns (Forsberg et al., 1988). In addition to evidence that strongly supports the role of C-fibres in reflex bronchoconstriction (Coleridge & Coleridge, 1986; Widdicombe, 1988) it has been demonstrated that activation of airway C-fibres causes the release of neuropeptides from the peripheral afferent nerve endings in the lung via a possible local axon reflex mechanism. These neuropeptides can cause bronchoconstriction, mucus secretion and oedema. Substance P (SP), was until recently, the only neuropeptide evident in the lung, known to evoke these responses (Lundberg & Saria, 1982), but biochemical and immunohistochemical evidence shows that many neuropeptides such as calcitonin gene-related peptide (CGRP) (Gibbins et al., 1985), neurokinin A, neurokinin B and neuropeptide K coexist with SP within the C-fibres (Hua et al., 1985). Much of the evidence which supports the role of C-fibres in airway reflexes comes from studies using capsaicin, the purported 'selective' C-fibre stimulant. Administration of capsaicin produces responses which are species-related. Intravenous or intra-arterial administration of capsaicin induces a predominantly non-cholinergic bronchoconstriction in the guinea-pig (Lundberg & Saria, 1982; Biggs & Goel, 1985) which is not affected by vagotomy. In contrast, in dog and cat, intravenous administration of capsaicin provokes bronchoconstriction which is dependent on a vagal cholinergic reflex (Russell & Lai Fook, 1979; Adcock, 1989). Aerosol adminis-
tration of capsaicin in cats also evokes a vagally-mediated cholinergic reflex bronchoconstriction, with no apparent noncholinergic component (Adcock & Smith, 1989), whilst in man, inhaled capsaicin causes cough and a transient, mainly cholinergic, bronchoconstriction (Fuller et al., 1985). It has been shown that in both vagal C-fibres and Ab-fibres innervating the respiratory tract of the cat, impulse activity was attenuated by intravenous infusion of the opioid peptide BW443C81 (Adcock et al., 1987, Adcock & Smith, 1987). In addition, in cats, bronchoconstrictor responses evoked by inhalation of capsaicin were attenuated by both intravenous and aerosolized BW443C81 (Adcock & Smith, 1989). The present study was designed to elucidate the mechanism by which capsaicin evokes bronchoconstriction in guinea-pigs when administered by aerosol. This was carried out by comparing the effects of atropine, bilateral vagotomy and the BW443C81 peripherally-acting opioid peptide (Tyr.D.Ala.Gly.Phe(4-NO2).Pro.NH2 diacetate) on the bronchoconstrictor responses evoked by inhaled capsaicin. Since codeine is a drug of choice in the clinical treatment of cough, results with this 'classical' opiate are included for comparison with BW443C81. A preliminary account of this work has been presented to the British Pharmacological Society (Buchan et al., 1989).
Methods Male Dunkin Hartley guinea-pigs (400-450 g) were anaesthetized with 2% halothane in oxygen at a flow rate of 2 1 min- 1. The right external jugular vein was cannulated with pp5O tubing (Portex) and anaesthesia was subsequently maintained by the intravenous administration of chloralose (100-
449
CAPSAICIN-INDUCED BRONCHOCONSTRICTION IN THE GUINEA-PIG
150mgkg-1; 10mlkg-'). Although it has been shown that halothane can stimulate/sensitize lung C-fibre endings (Coleridge & Coleridge, 1984), in our laboratory this effect on C-fibres is short lived in anaesthetized cats. Further, baseline airway tone is no different in guinea-pigs anaesthetized with halothane compared to animals anaesthetized with barbiturate (unpublished observations). The left external jugular vein was cannulated with pp6O tubing (Portex) for bolus administration of atropine or infusion of either BW443C81 or codeine. The trachea was cannulated and animals were artificially ventilated at a rate of 50 breaths min' with laboratory air, 1O ml kg- l (Palmer Bioscience ventilator). With this ventilation regime blood gases and pH were satisfactorily maintained at physiological levels. Pulmonary inflation pressure (PIP) was measured with a Statham P23 pressure transducer attached to a side-arm of the tracheal cannula. The left carotid artery was cannulated with pp6O tubing (Portex) containing heparinised saline (125 u ml-'), to record arterial blood pressure (Statham P23 transducer) and heart rate was derived from the arterial pulse with a cardiotachometer. Cardiovascular and pulmonary variables were recorded continuously on a Beckman R611 recorder. Body temperature was maintained at 37-380C with a heated operating table.
Experimental protocol Animals were allowed to stabilize for a minimum of 20 min before administration of aerosol. Capsaicin, (5 ml, 0.9 ug ml- 1) was added to the well of a modified DeVilbiss ultrasonic nebuliser (Lees & Payne, 1986) placed in the air inflow circuit from the ventilator. Each animal received separate administrations of capsaicin 5, 10 and 15 breaths. In some experiments, atropine (0.3mgkg-1), was administered as an intravenous bolus, 10min prior to the first capsaicin inhalation. This dose of atropine caused complete abolition of the bronchoconstrictor response to intravenous administration of acetylcholine (lOpgkg-1) for the duration of the experiment of approximately 60min. In other experiments, before administration of capsaicin, bilateral vagotomy was performed by sectioning the vago-sympathetic bundles. BW443C81 (3, 30, lOOpgkg-'min-'), codeine or N-methyl nalorphine at lOOpgkg-'min-m were administered by intravenous infusion, which started 10min before capsaicin administration was begun. The total administered dose of BW443C81 and codeine at lOOpgkg-'min-1, was 6mgkg-' over the 1 h infusion period. This protocol minimized the cardiovascular effects of the two drugs and maintained effective BW443C81 plasma concentrations (Adcock, 1989). A peristaltic cassette pump (202U Watson Marlow) was used to infuse drugs at a rate of 0.1 ml min -
tration. The duration of the bronchoconstrictor response, especially at 15 breaths, was quite prolonged and thus the area of response was more representative of capsaicin action rather than the magnitude or peak height of response. The areas were calculated using an Apple HIe computer with graphics tablet. Statistical significance of drug treatments was determined by comparing mean difference in areas (A areas) of each capsaicin response in drug-treated animals with those in saline-treated animals by Student's t test for unpaired data.
Drugs Drugs and chemicals were obtained from the following sources: BW443C81, synthesized by Dr L.A. Lowe and Nmethyl nalorphine, synthesized by the late Dr S. Wilkinson (Wellcome Research Laboratories); codeine phosphate (Wellcome Research Laboratories); atropine sulphate (BDH Chemicals Ltd); a-chloralose (Koch-Light Ltd); capsaicin (Fluka AG); ethanol (BDH Chemicals Ltd); Euthesate and Halothane (May & Baker Ltd); heparin (Evans Medical Ltd); Tween 80 (Sigma). Capsaicin was dissolved in an ethanol (0.2 ml), Tween 80 (0.2 ml) and saline (9.6 ml) mixture to 1 mg ml - ' and diluted with saline to 0.9 ug ml 1 of free base. All other drugs were dissolved in saline and doses are expressed as free base.
Results
Effect of inhalation of capsaicin Inhalation of aerosols of the vehicle for capsaicin had no effect on the cardiopulmonary variables measured in these experiments. In preliminary experiments (results not shown) tachyphylaxis developed to bronchoconstrictor effects of capsaicin aerosol if either multiple dose-response curves were attempted or large doses were administered repeatedly. Inhalation from a 0.3 pg ml'- solution of capsaicin, in most animals, produced a threshold response whilst 3.OpgmlP1 produced respiratory and cardiovascular distress. Therefore, only one dose-response curve was carried out in each animal using 5, 10 and 15 breaths of a 0.9 pgml-' solution which produced a doserelated increase in PIP. This increase in PIP was completely reversed by 2 breath hyperinflations. A typical response to capsaicin (15 breaths), showing the extended duration of bronchoconstriction, is shown in Figure 1.
Effect of bilateral vagotomy and atropine Data analysis Bronchoconstrictor responses to capsaicin were determined by measurement of the change between areas of response (mm2) on the pulmonary inflation pressure (PIP) traces for a duration of 5 min before and 5 min after capsaicin adminis-
Capsaicin-induced bronchoconstrictor responses were significantly, but not totally inhibited by bilateral vagotomy (46% + 14% at 10 breaths capsaicin, P < 0.05, n = 13) or the administration of atropine 0.3 mgkg-' i.v. bolus (59% + 13% at 10 breaths capsaicin, P < 0.01, n = 14) (Figure 2).
Capsaicin
1 min
10 breaths
iiIEEEEI----
--m__ ...~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I
Predose 5 min
Postdose 5 min
Figure 1 A typical trace (pulmonary inflation pressure, PIP) of the bronchoconstrictor response evoked by inhalation of capsaicin (15 breaths, 0.9pjugml-') in an artificially ventilated anaesthetized guinea-pig.
450
P. BUCHAN & J.J. ADCOCK
2000r
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1500
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a 1000
1000
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15
Breaths of capsaicin (0.9 pug ml-')
Breaths of capsaicin (0.9 pLg ml') Figure 2 Bronchoconstrictor responses in artificially ventilated anaesthetized guinea-pigs following inhalation of capsaicin (5, 10, 15 breaths, 0.9pgmIP'): saline, 0.1mlmin-' (0. n = 20); atropine 0.3mgkg-', i.v. (El. n = 14); bilateral vagotomy (0, n = 13); BW443C81 301ugkg-1min-', i.v. (U, n = 6). The results are expressed as areas of response (postdose-predose) and represent mean with s.e.mean of areas (mm2) shown by vertical bars. Statistical significance between treated groups and control groups was determined by Student's t test for unpaired data and shown by * P < 0.05; ** P < 0.01 *** P < 0.005; BW443C81 compared to either atropine or vagotomy is shown by t P < 0.05; ttt P < 0.005.
I
10
Figure 4 Effect of N-methyl nalorphine (N-MeNal) on the inhibition by BW443C81 of bronchoconstrictor responses evoked by inhalation of capsaicin (5, 10, 15 breaths, 0.9,pgmlP ) in artificially ventilated anaesthetized guinea-pigs: N-MeNal 100,ugkg- min- , i.v. (0, n = 5); BW443C81 30upgkg-1min 1, i.v. (EO, n = 6) or BW443C81 30pgkg-1 min-, i.v. + N-MeNal 100pgkg-1min-1, i.v. (0, n = 6). The results are expressed as areas of response (postdose-predose) and represent mean with s.e.mean of areas (mm2) shown by vertical bars. Statistical significance between treated groups and control groups was determined by Student's t test for unpaired data and shown by *** P < 0.005; N-MeNal compared to BW443C81 is shown by tP
C.) Macmillan Press Ltd, 1992
Capsaicin-induced bronchoconstriction in the guinea-pig: contribution of vagal cholinergic reflexes, local axon reflexes and their modulation by BW443C81 P. Buchan & J.J. Adcock Department of Pharmacology, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS 1 The objective of the study was to investigate the central vagal and local axon reflex components of bronchoconstrictor responses evoked by inhalation of capsaicin aerosol in anaesthetized guinea-pigs. This was accomplished by comparing the effects of bilateral vagotomy, atropine and the peripherally-acting polar enkephalin analogue, BW443C81, on bronchoconstrictor responses evoked by capsaicin. The effects of codeine were also determined. 2 Aerosols of capsaicin were generated from a 0.9 pg ml-' solution. Inhalation of capsaicin aerosol in 5, 10 and 15 breaths evoked dose-related bronchoconstrictor responses. The responses were immediate in onset and of extended duration. 3 Capsaicin-induced bronchoconstrictor responses were significantly inhibited following bilateral vagotomy or atropine (0.3 mgkg-1, i.v.) pretreatment by 46% + 14% (P < 0.05) and 59% + 13% (P < 0.01), respectively. 4 Administration of BW443C81 by intravenous infusion (3, 30 and lOOpgkg- min-1) caused a significant inhibition of capsaicin-induced bronchoconstrictor responses which achieved a greater maximum than either bilateral vagotomy or atropine. Codeine (l00 pgkg-'min-t, i.v.) did not significantly inhibit the bronchoconstrictor responses. 5 Inhibition of capsaicin-induced bronchoconstrictor responses by BW443C81 (30pgkg-1min-1, i.v.) was significantly (P < 0.05) reduced by the peripherally-acting opioid antagonist N-methyl nalorphine (l00pgkg- min- i.v.). 6 These results show that capsaicin-induced bronchoconstrictor responses are mediated by at least two mechanisms, a vagal and/or cholinergic reflex pathway and a non-cholinergic pathway. BW443C81, but not codeine, significantly inhibited (P < 0.005) both mechanisms of capsaicin-induced bronchoconstriction probably by an action on peripheral opioid receptors located on vagal sensory nerves. Keywords: Reflex bronchoconstriction; inhalation; capsaicin; sensory nerves ,
Introduction Stimulation of C-fibre afferents in the lung may contribute to a variety of airway reflexes including cough (Winning et al., 1986; Karlsson et al., 1988; Fuller et al., 1988), bronchoconstriction (Forsberg & Karlsson, 1986), mucus secretion (Davis et al., 1982) as well as changes in breathing patterns (Forsberg et al., 1988). In addition to evidence that strongly supports the role of C-fibres in reflex bronchoconstriction (Coleridge & Coleridge, 1986; Widdicombe, 1988) it has been demonstrated that activation of airway C-fibres causes the release of neuropeptides from the peripheral afferent nerve endings in the lung via a possible local axon reflex mechanism. These neuropeptides can cause bronchoconstriction, mucus secretion and oedema. Substance P (SP), was until recently, the only neuropeptide evident in the lung, known to evoke these responses (Lundberg & Saria, 1982), but biochemical and immunohistochemical evidence shows that many neuropeptides such as calcitonin gene-related peptide (CGRP) (Gibbins et al., 1985), neurokinin A, neurokinin B and neuropeptide K coexist with SP within the C-fibres (Hua et al., 1985). Much of the evidence which supports the role of C-fibres in airway reflexes comes from studies using capsaicin, the purported 'selective' C-fibre stimulant. Administration of capsaicin produces responses which are species-related. Intravenous or intra-arterial administration of capsaicin induces a predominantly non-cholinergic bronchoconstriction in the guinea-pig (Lundberg & Saria, 1982; Biggs & Goel, 1985) which is not affected by vagotomy. In contrast, in dog and cat, intravenous administration of capsaicin provokes bronchoconstriction which is dependent on a vagal cholinergic reflex (Russell & Lai Fook, 1979; Adcock, 1989). Aerosol adminis-
tration of capsaicin in cats also evokes a vagally-mediated cholinergic reflex bronchoconstriction, with no apparent noncholinergic component (Adcock & Smith, 1989), whilst in man, inhaled capsaicin causes cough and a transient, mainly cholinergic, bronchoconstriction (Fuller et al., 1985). It has been shown that in both vagal C-fibres and Ab-fibres innervating the respiratory tract of the cat, impulse activity was attenuated by intravenous infusion of the opioid peptide BW443C81 (Adcock et al., 1987, Adcock & Smith, 1987). In addition, in cats, bronchoconstrictor responses evoked by inhalation of capsaicin were attenuated by both intravenous and aerosolized BW443C81 (Adcock & Smith, 1989). The present study was designed to elucidate the mechanism by which capsaicin evokes bronchoconstriction in guinea-pigs when administered by aerosol. This was carried out by comparing the effects of atropine, bilateral vagotomy and the BW443C81 peripherally-acting opioid peptide (Tyr.D.Ala.Gly.Phe(4-NO2).Pro.NH2 diacetate) on the bronchoconstrictor responses evoked by inhaled capsaicin. Since codeine is a drug of choice in the clinical treatment of cough, results with this 'classical' opiate are included for comparison with BW443C81. A preliminary account of this work has been presented to the British Pharmacological Society (Buchan et al., 1989).
Methods Male Dunkin Hartley guinea-pigs (400-450 g) were anaesthetized with 2% halothane in oxygen at a flow rate of 2 1 min- 1. The right external jugular vein was cannulated with pp5O tubing (Portex) and anaesthesia was subsequently maintained by the intravenous administration of chloralose (100-
449
CAPSAICIN-INDUCED BRONCHOCONSTRICTION IN THE GUINEA-PIG
150mgkg-1; 10mlkg-'). Although it has been shown that halothane can stimulate/sensitize lung C-fibre endings (Coleridge & Coleridge, 1984), in our laboratory this effect on C-fibres is short lived in anaesthetized cats. Further, baseline airway tone is no different in guinea-pigs anaesthetized with halothane compared to animals anaesthetized with barbiturate (unpublished observations). The left external jugular vein was cannulated with pp6O tubing (Portex) for bolus administration of atropine or infusion of either BW443C81 or codeine. The trachea was cannulated and animals were artificially ventilated at a rate of 50 breaths min' with laboratory air, 1O ml kg- l (Palmer Bioscience ventilator). With this ventilation regime blood gases and pH were satisfactorily maintained at physiological levels. Pulmonary inflation pressure (PIP) was measured with a Statham P23 pressure transducer attached to a side-arm of the tracheal cannula. The left carotid artery was cannulated with pp6O tubing (Portex) containing heparinised saline (125 u ml-'), to record arterial blood pressure (Statham P23 transducer) and heart rate was derived from the arterial pulse with a cardiotachometer. Cardiovascular and pulmonary variables were recorded continuously on a Beckman R611 recorder. Body temperature was maintained at 37-380C with a heated operating table.
Experimental protocol Animals were allowed to stabilize for a minimum of 20 min before administration of aerosol. Capsaicin, (5 ml, 0.9 ug ml- 1) was added to the well of a modified DeVilbiss ultrasonic nebuliser (Lees & Payne, 1986) placed in the air inflow circuit from the ventilator. Each animal received separate administrations of capsaicin 5, 10 and 15 breaths. In some experiments, atropine (0.3mgkg-1), was administered as an intravenous bolus, 10min prior to the first capsaicin inhalation. This dose of atropine caused complete abolition of the bronchoconstrictor response to intravenous administration of acetylcholine (lOpgkg-1) for the duration of the experiment of approximately 60min. In other experiments, before administration of capsaicin, bilateral vagotomy was performed by sectioning the vago-sympathetic bundles. BW443C81 (3, 30, lOOpgkg-'min-'), codeine or N-methyl nalorphine at lOOpgkg-'min-m were administered by intravenous infusion, which started 10min before capsaicin administration was begun. The total administered dose of BW443C81 and codeine at lOOpgkg-'min-1, was 6mgkg-' over the 1 h infusion period. This protocol minimized the cardiovascular effects of the two drugs and maintained effective BW443C81 plasma concentrations (Adcock, 1989). A peristaltic cassette pump (202U Watson Marlow) was used to infuse drugs at a rate of 0.1 ml min -
tration. The duration of the bronchoconstrictor response, especially at 15 breaths, was quite prolonged and thus the area of response was more representative of capsaicin action rather than the magnitude or peak height of response. The areas were calculated using an Apple HIe computer with graphics tablet. Statistical significance of drug treatments was determined by comparing mean difference in areas (A areas) of each capsaicin response in drug-treated animals with those in saline-treated animals by Student's t test for unpaired data.
Drugs Drugs and chemicals were obtained from the following sources: BW443C81, synthesized by Dr L.A. Lowe and Nmethyl nalorphine, synthesized by the late Dr S. Wilkinson (Wellcome Research Laboratories); codeine phosphate (Wellcome Research Laboratories); atropine sulphate (BDH Chemicals Ltd); a-chloralose (Koch-Light Ltd); capsaicin (Fluka AG); ethanol (BDH Chemicals Ltd); Euthesate and Halothane (May & Baker Ltd); heparin (Evans Medical Ltd); Tween 80 (Sigma). Capsaicin was dissolved in an ethanol (0.2 ml), Tween 80 (0.2 ml) and saline (9.6 ml) mixture to 1 mg ml - ' and diluted with saline to 0.9 ug ml 1 of free base. All other drugs were dissolved in saline and doses are expressed as free base.
Results
Effect of inhalation of capsaicin Inhalation of aerosols of the vehicle for capsaicin had no effect on the cardiopulmonary variables measured in these experiments. In preliminary experiments (results not shown) tachyphylaxis developed to bronchoconstrictor effects of capsaicin aerosol if either multiple dose-response curves were attempted or large doses were administered repeatedly. Inhalation from a 0.3 pg ml'- solution of capsaicin, in most animals, produced a threshold response whilst 3.OpgmlP1 produced respiratory and cardiovascular distress. Therefore, only one dose-response curve was carried out in each animal using 5, 10 and 15 breaths of a 0.9 pgml-' solution which produced a doserelated increase in PIP. This increase in PIP was completely reversed by 2 breath hyperinflations. A typical response to capsaicin (15 breaths), showing the extended duration of bronchoconstriction, is shown in Figure 1.
Effect of bilateral vagotomy and atropine Data analysis Bronchoconstrictor responses to capsaicin were determined by measurement of the change between areas of response (mm2) on the pulmonary inflation pressure (PIP) traces for a duration of 5 min before and 5 min after capsaicin adminis-
Capsaicin-induced bronchoconstrictor responses were significantly, but not totally inhibited by bilateral vagotomy (46% + 14% at 10 breaths capsaicin, P < 0.05, n = 13) or the administration of atropine 0.3 mgkg-' i.v. bolus (59% + 13% at 10 breaths capsaicin, P < 0.01, n = 14) (Figure 2).
Capsaicin
1 min
10 breaths
iiIEEEEI----
--m__ ...~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I
Predose 5 min
Postdose 5 min
Figure 1 A typical trace (pulmonary inflation pressure, PIP) of the bronchoconstrictor response evoked by inhalation of capsaicin (15 breaths, 0.9pjugml-') in an artificially ventilated anaesthetized guinea-pig.
450
P. BUCHAN & J.J. ADCOCK
2000r
-
g- 1500 E E
1500
E E
ttt
a)
a1)
L-
a 1000
1000
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CoC 0. C5
0.
0
cr
c
500
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500
IF IN
5
15
Breaths of capsaicin (0.9 pug ml-')
Breaths of capsaicin (0.9 pLg ml') Figure 2 Bronchoconstrictor responses in artificially ventilated anaesthetized guinea-pigs following inhalation of capsaicin (5, 10, 15 breaths, 0.9pgmIP'): saline, 0.1mlmin-' (0. n = 20); atropine 0.3mgkg-', i.v. (El. n = 14); bilateral vagotomy (0, n = 13); BW443C81 301ugkg-1min-', i.v. (U, n = 6). The results are expressed as areas of response (postdose-predose) and represent mean with s.e.mean of areas (mm2) shown by vertical bars. Statistical significance between treated groups and control groups was determined by Student's t test for unpaired data and shown by * P < 0.05; ** P < 0.01 *** P < 0.005; BW443C81 compared to either atropine or vagotomy is shown by t P < 0.05; ttt P < 0.005.
I
10
Figure 4 Effect of N-methyl nalorphine (N-MeNal) on the inhibition by BW443C81 of bronchoconstrictor responses evoked by inhalation of capsaicin (5, 10, 15 breaths, 0.9,pgmlP ) in artificially ventilated anaesthetized guinea-pigs: N-MeNal 100,ugkg- min- , i.v. (0, n = 5); BW443C81 30upgkg-1min 1, i.v. (EO, n = 6) or BW443C81 30pgkg-1 min-, i.v. + N-MeNal 100pgkg-1min-1, i.v. (0, n = 6). The results are expressed as areas of response (postdose-predose) and represent mean with s.e.mean of areas (mm2) shown by vertical bars. Statistical significance between treated groups and control groups was determined by Student's t test for unpaired data and shown by *** P < 0.005; N-MeNal compared to BW443C81 is shown by tP
