Inhibitory effects of catecholamines in isolated canine bronchial smooth P. A. VERMEIRE AND Department of Medicine,
P. M. VANHOUTTE Universitaire Instelling
VERMEIRE, P. A., AND P. M. VANHOUTE. Inhibitory effects of catecholamines in isolated canine bronchiaL smooth muscle. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 46(d): 787-791, 1979.-Experiments were designed to investigate whether catecholamines affect cholinergic neurotransmission in the bronchial wall. Rings of canine bronchi were mounted for continuous isometric tension recording. Acetylcholine and electrical stimulation caused contractions. As the response to electrical stimulation was unaffected by hexamethonium; but inhibited by tetrodotoxin and atropine, it must be due to release of endogenous acetylcholine form postganglionic cholinergic nerves. Norepinephrine and isoproterenol inhibited the response to electrical stimulation significantly more than that to acetylcholine; propranolol abolished the inhibition in both cases. These findings suggest that in the canine bronchi catecholamines induce dilation during bronchoconstriction caused by increased cholinergic nerve activity, whereas they have relatively weak inhibitory effects on bronchial smooth muscle made to contract with exogenous acetylcholine. Propranolol augmented the contractile response to electrical stimulation, but not that to acetylcholine. Helical bronchial strips were incubated with [3H] norepinephrine and mounted for superfusion; the overflow of tritiated compounds in the superfusate was measured. Electrical stimulation augmented markedly the “H efflux. These observations indicate that the electric impulses activate both cholinergic and adrenergic nerves in the bronchial wall, and that endogenously released norepinephrine can partially inhibit bronchoconstrictions caused by stimulation of the cholinergic nerves. adrenergic nerves in bronchial wall; airway smooth muscle; atropine; bronchial rings; bronchodilatation; cholinergic nerves in bronchial wall; choline@ neuroeffector interaction; isoproterenol; local autonomic modulation; norepinephrine; [“HInorepinephrine; propranolol; tetrodotoxin
BRONCHIAL HYPERRESPONSIVENESS, a major feature Of bronchial asthma, is most likely the consequence of a functional imbalance between cholinergic and adrenergic control of airway smooth muscle (1, 9, 14, 16, 18, 21). Although most studies on tracheal and bronchial preparations have focused on the interaction between the adrenergic and choline@ neurotransmitters at the level of the smooth muscle cells, little is known of the action of catecholamines on the cholinergic nerve terminals in the bronchial wall (3, 6, 11, 14, 19). Therefore, experiments were designed to investigate the effect of exogenously added and endogenously released catecholamines on the response of isolated canine bronchi to cholinergic activation. 0161-7567/79/0000-oooO$O1.25
Copyright
0 1979 the American
Physiological
Antwerpen,
muscle
2610
Wilrijk, Belgium
METHODS
The experiments were performed on rings (width 4 mm) of intrapulmonary lobar or segmental bronchi (ID 5 mm) taken from dogs (15-25 kg) anesthetized with pentobarbital (30 mg/kg i.v.). The preparations were mounted in an organ chamber filed with Krebs-Ringer bicarbonate solution of the following millimolar composition: NaCl, 118.3; KCl, 4.7; MgSO,, 1.2; KHzPOd, 1.2; CaC12,2.5; NaHC03, 25; calcium disodium ethylenediaminetetraacetate, 0.026; and glucose, 11.1. The solution (pH 7.4) was maintained at 37OC and continuously aerated with a mixture of 95% 02 and 5% COZ. The rings were connected to a strain gauge (Grass FT 03) for continuous isometric tension recording. When drugs were added to the bath solution, they were contained in 0.1 ml of distilled water. For electrical stimulation of the preparations, two rectangular platinum electrodes were placed parallel to the rings; electric impulses consisted of square waves (9 V, 2 ms) provided by a DC power supply and switching transistor (Siemens, AD 149) triggered by a stimulator (Grass S88). Before the experiments were begun, the preparations were placed at the optimal point of their length-tension relationship, using a standard electrical stimulation (15 Hz for 10 s); the rings were allowed to equilibrate at their optimal length for 60 min prior to experimentation. Drugs. The following pharmacological agents were used: acetylcholine chloride (Sigma, St. Louis, MO), atropine sulphate (Merck, Darmstadt, W. Germany), hexamethonium bromide (Sigma), isoproterenol hydrochloride (Aldrich Europe, Beerse, Belgium), I-norepinephrine d-bitartrate (Fluka AG, Buchs, Switzerland), propranolol hydrochloride (I.C.I. Pharma, Gent, Belgium), and tetrodotoxin (Sigma). rH]norepinephrine efflux. In certain experiments, helical bronchial strips (width 4 mm, length 5 cm) were incubated for 4 h in Krebs-Ringer solution containing 3 x low7 M [7-“Hlnorepinephrine (sp act 8.8 Ci/mmol, Amersham). At the end of the incubation period the strips were rinsed in fresh Krebs-Ringer solution and mounted for superfusion (23, 26, 27). They were suspended in a moist tunnel-shaped chamber maintained at 37°C and were superfused at 3 ml/min by a constantflow roller pump with Krebs-Ringer solution previously aerated with 95% 02 and 5% COZ. Acetylcholine was added to the superfusate upstream from the roller pump. For electrical stimulation of the preparations, two platinum wires (0.5 mm in diam) were placed parallel to the Society
787
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 16, 2019.
788
P. A. VERMEIRE
bronchi. Both the strips and the electrodes were continuously superfused. The preparations were connected to a strain gauge for isometric tension recording. The initial tension was set at 3 g. After this initial stretch, the tension decreased and stabilized within 30 min. At this time sampling of the superfusate was started at 2-min intervals for direct estimation of total radioactivity. Aliquots (1 ml) of superfusate were added to 10 ml of InstaGel (Packard Instrument), and the radioactivity was measured in a liquid scintillation counter. Corrections for quenching were made with an external standard. The counting efficiency was 42%. The samples were counted for 10 min. Statistical analysis. For each group of preparations the number reported is also the number of dogs used. The data are expressed as means tSE. For the statistical analysis of the data, Student’s t test for paired and unpaired observations was used. P values smaller than 0.05 were considered to be significant. RESULTS
Paired bronchial rings from six dogs were exposed to electrical stimulation at increasing frequencies (0.5-30 Hz) and to acetylcholine in increasing concentrations ( 10e7to 10e3M), respectively (Fig. 1). Both stimuli caused contraction of the isolated bronchi; the maximal response to acetylcholine was significantly greater than that to electrical stimulation. From these preliminary data, a stimulation frequency (15 Hz) and an acetylcholine concentration (lo-” M), which caused comparable contractile responses, were selected for further experimentation; for the 39 pairs of rings reported the average increase in tension obtained with electrical stimulation was 2.73 t 0.46 g, and with acetylcholine 3.44 t 0.52 g. ControZ responses. Two groups of six bronchial rings each were made to contract with 15 Hz and 10m5M acetylcholine, respectively. The stimulus was applied for
% E
ELECTRIC
ACETYLCHOLINE
STIMULATION
G r
80 E
2 2
AND
P. M.
VANHOUTE
l-3 min (until a peak response was obtained) at 15-min intervals for 3 h; after each contraction the preparations were washed with fresh Krebs-Ringer. The response to both electrical stimulation and acetylcholine was reproducible over the time period tested (Table 1). Hexamethonium. In five pairs of bronchial rings, the effect of 5 x lo-” M hexamethonium was investigated on the contractile response to electrical stimulation and acetylcholine, respectively. After incubation with the ganglionic blocking drug the response to electrical stimulation averaged 95.9 t 8.8% of control, and that to acetylcholine 104.1 t 2.0%; these differences from control were not significant. Tetrodotoxin. In four bronchial rings, tetrodotoxin (3 x 10SHM) significantly reduced the response to electrical stimulation to 2.0 t 1.2% of the control reaction; by contrast in paired rings the contractile response to acetylcholine in presence of tetrodotoxin averaged 110.2 t 9.2% of the control increase in tension. The difference in effect of tetrodotoxin on the response to electrical stimulation and acetylcholine was statistically significant. Atropine. In six paired preparations, the effect of increasing concentrations of atropine (lo-“’ to 10m7M) was investigated on the response to electrical stimulation and acetylcholine, respectively (Fig. 2). In both cases, atropine caused a dose-dependent inhibition of the contractile responses. Significant depressions were seen with concentrations of atropine as low as 5 x lo-” M; at 10D7 M atropine the response to both types of stimuli was abolished. CatechoZamines. The effects of increasing concentrations of norepinephrine (10B8to 10S4M) and isoprotereno1 (10e8 to 10e4 M) were investigated in two groups of six paired preparations. At the concentrations used the catecholamines did not cause significant changes in basal tension. Both norepinephrine and isoproterenol caused a dose-dependent inhibition of the response to electrical stimulation (Fig. 3); the inhibition was significant from concentrations of lo+ M norepinephrine and 10B7 M isoproterenol on. Both catecholamines also inhibited the response to exogenous acetylcholine but a significant inhibition was only obtained with isoproterenol from concentrations of low6 M on. With all concentrations of norepinephrine, and with lo-” to lOa M isoproterenol, the inhibition of the response to electrical stimulation 1. Responses to electrical stimulation and acetylcholine as a function of time -- ------ -- -__----
k
TABLE
P. M. VANHOUTTE Universitaire Instelling
VERMEIRE, P. A., AND P. M. VANHOUTE. Inhibitory effects of catecholamines in isolated canine bronchiaL smooth muscle. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 46(d): 787-791, 1979.-Experiments were designed to investigate whether catecholamines affect cholinergic neurotransmission in the bronchial wall. Rings of canine bronchi were mounted for continuous isometric tension recording. Acetylcholine and electrical stimulation caused contractions. As the response to electrical stimulation was unaffected by hexamethonium; but inhibited by tetrodotoxin and atropine, it must be due to release of endogenous acetylcholine form postganglionic cholinergic nerves. Norepinephrine and isoproterenol inhibited the response to electrical stimulation significantly more than that to acetylcholine; propranolol abolished the inhibition in both cases. These findings suggest that in the canine bronchi catecholamines induce dilation during bronchoconstriction caused by increased cholinergic nerve activity, whereas they have relatively weak inhibitory effects on bronchial smooth muscle made to contract with exogenous acetylcholine. Propranolol augmented the contractile response to electrical stimulation, but not that to acetylcholine. Helical bronchial strips were incubated with [3H] norepinephrine and mounted for superfusion; the overflow of tritiated compounds in the superfusate was measured. Electrical stimulation augmented markedly the “H efflux. These observations indicate that the electric impulses activate both cholinergic and adrenergic nerves in the bronchial wall, and that endogenously released norepinephrine can partially inhibit bronchoconstrictions caused by stimulation of the cholinergic nerves. adrenergic nerves in bronchial wall; airway smooth muscle; atropine; bronchial rings; bronchodilatation; cholinergic nerves in bronchial wall; choline@ neuroeffector interaction; isoproterenol; local autonomic modulation; norepinephrine; [“HInorepinephrine; propranolol; tetrodotoxin
BRONCHIAL HYPERRESPONSIVENESS, a major feature Of bronchial asthma, is most likely the consequence of a functional imbalance between cholinergic and adrenergic control of airway smooth muscle (1, 9, 14, 16, 18, 21). Although most studies on tracheal and bronchial preparations have focused on the interaction between the adrenergic and choline@ neurotransmitters at the level of the smooth muscle cells, little is known of the action of catecholamines on the cholinergic nerve terminals in the bronchial wall (3, 6, 11, 14, 19). Therefore, experiments were designed to investigate the effect of exogenously added and endogenously released catecholamines on the response of isolated canine bronchi to cholinergic activation. 0161-7567/79/0000-oooO$O1.25
Copyright
0 1979 the American
Physiological
Antwerpen,
muscle
2610
Wilrijk, Belgium
METHODS
The experiments were performed on rings (width 4 mm) of intrapulmonary lobar or segmental bronchi (ID 5 mm) taken from dogs (15-25 kg) anesthetized with pentobarbital (30 mg/kg i.v.). The preparations were mounted in an organ chamber filed with Krebs-Ringer bicarbonate solution of the following millimolar composition: NaCl, 118.3; KCl, 4.7; MgSO,, 1.2; KHzPOd, 1.2; CaC12,2.5; NaHC03, 25; calcium disodium ethylenediaminetetraacetate, 0.026; and glucose, 11.1. The solution (pH 7.4) was maintained at 37OC and continuously aerated with a mixture of 95% 02 and 5% COZ. The rings were connected to a strain gauge (Grass FT 03) for continuous isometric tension recording. When drugs were added to the bath solution, they were contained in 0.1 ml of distilled water. For electrical stimulation of the preparations, two rectangular platinum electrodes were placed parallel to the rings; electric impulses consisted of square waves (9 V, 2 ms) provided by a DC power supply and switching transistor (Siemens, AD 149) triggered by a stimulator (Grass S88). Before the experiments were begun, the preparations were placed at the optimal point of their length-tension relationship, using a standard electrical stimulation (15 Hz for 10 s); the rings were allowed to equilibrate at their optimal length for 60 min prior to experimentation. Drugs. The following pharmacological agents were used: acetylcholine chloride (Sigma, St. Louis, MO), atropine sulphate (Merck, Darmstadt, W. Germany), hexamethonium bromide (Sigma), isoproterenol hydrochloride (Aldrich Europe, Beerse, Belgium), I-norepinephrine d-bitartrate (Fluka AG, Buchs, Switzerland), propranolol hydrochloride (I.C.I. Pharma, Gent, Belgium), and tetrodotoxin (Sigma). rH]norepinephrine efflux. In certain experiments, helical bronchial strips (width 4 mm, length 5 cm) were incubated for 4 h in Krebs-Ringer solution containing 3 x low7 M [7-“Hlnorepinephrine (sp act 8.8 Ci/mmol, Amersham). At the end of the incubation period the strips were rinsed in fresh Krebs-Ringer solution and mounted for superfusion (23, 26, 27). They were suspended in a moist tunnel-shaped chamber maintained at 37°C and were superfused at 3 ml/min by a constantflow roller pump with Krebs-Ringer solution previously aerated with 95% 02 and 5% COZ. Acetylcholine was added to the superfusate upstream from the roller pump. For electrical stimulation of the preparations, two platinum wires (0.5 mm in diam) were placed parallel to the Society
787
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 16, 2019.
788
P. A. VERMEIRE
bronchi. Both the strips and the electrodes were continuously superfused. The preparations were connected to a strain gauge for isometric tension recording. The initial tension was set at 3 g. After this initial stretch, the tension decreased and stabilized within 30 min. At this time sampling of the superfusate was started at 2-min intervals for direct estimation of total radioactivity. Aliquots (1 ml) of superfusate were added to 10 ml of InstaGel (Packard Instrument), and the radioactivity was measured in a liquid scintillation counter. Corrections for quenching were made with an external standard. The counting efficiency was 42%. The samples were counted for 10 min. Statistical analysis. For each group of preparations the number reported is also the number of dogs used. The data are expressed as means tSE. For the statistical analysis of the data, Student’s t test for paired and unpaired observations was used. P values smaller than 0.05 were considered to be significant. RESULTS
Paired bronchial rings from six dogs were exposed to electrical stimulation at increasing frequencies (0.5-30 Hz) and to acetylcholine in increasing concentrations ( 10e7to 10e3M), respectively (Fig. 1). Both stimuli caused contraction of the isolated bronchi; the maximal response to acetylcholine was significantly greater than that to electrical stimulation. From these preliminary data, a stimulation frequency (15 Hz) and an acetylcholine concentration (lo-” M), which caused comparable contractile responses, were selected for further experimentation; for the 39 pairs of rings reported the average increase in tension obtained with electrical stimulation was 2.73 t 0.46 g, and with acetylcholine 3.44 t 0.52 g. ControZ responses. Two groups of six bronchial rings each were made to contract with 15 Hz and 10m5M acetylcholine, respectively. The stimulus was applied for
% E
ELECTRIC
ACETYLCHOLINE
STIMULATION
G r
80 E
2 2
AND
P. M.
VANHOUTE
l-3 min (until a peak response was obtained) at 15-min intervals for 3 h; after each contraction the preparations were washed with fresh Krebs-Ringer. The response to both electrical stimulation and acetylcholine was reproducible over the time period tested (Table 1). Hexamethonium. In five pairs of bronchial rings, the effect of 5 x lo-” M hexamethonium was investigated on the contractile response to electrical stimulation and acetylcholine, respectively. After incubation with the ganglionic blocking drug the response to electrical stimulation averaged 95.9 t 8.8% of control, and that to acetylcholine 104.1 t 2.0%; these differences from control were not significant. Tetrodotoxin. In four bronchial rings, tetrodotoxin (3 x 10SHM) significantly reduced the response to electrical stimulation to 2.0 t 1.2% of the control reaction; by contrast in paired rings the contractile response to acetylcholine in presence of tetrodotoxin averaged 110.2 t 9.2% of the control increase in tension. The difference in effect of tetrodotoxin on the response to electrical stimulation and acetylcholine was statistically significant. Atropine. In six paired preparations, the effect of increasing concentrations of atropine (lo-“’ to 10m7M) was investigated on the response to electrical stimulation and acetylcholine, respectively (Fig. 2). In both cases, atropine caused a dose-dependent inhibition of the contractile responses. Significant depressions were seen with concentrations of atropine as low as 5 x lo-” M; at 10D7 M atropine the response to both types of stimuli was abolished. CatechoZamines. The effects of increasing concentrations of norepinephrine (10B8to 10S4M) and isoprotereno1 (10e8 to 10e4 M) were investigated in two groups of six paired preparations. At the concentrations used the catecholamines did not cause significant changes in basal tension. Both norepinephrine and isoproterenol caused a dose-dependent inhibition of the response to electrical stimulation (Fig. 3); the inhibition was significant from concentrations of lo+ M norepinephrine and 10B7 M isoproterenol on. Both catecholamines also inhibited the response to exogenous acetylcholine but a significant inhibition was only obtained with isoproterenol from concentrations of low6 M on. With all concentrations of norepinephrine, and with lo-” to lOa M isoproterenol, the inhibition of the response to electrical stimulation 1. Responses to electrical stimulation and acetylcholine as a function of time -- ------ -- -__----
k
TABLE
