Agents Actions 36 (1992)
0065-4299/92/020029-04 $1.50 + 0.20/0 9 1992 Birkhfiuser Verlag, Basel
Modulation of neurally mediated airway microvascular leakage in guinea-pig airways by fl2-adrenoceptor agonists K. P. Hui, P. Ventresca, A. C. Brown 1, p. j. Barnes and K. F. Chung Department of Thoracic Medicine, National Heart & Lung Institute, Royal Brompton National Heart & Lung Hospital, London, SW3, UK and i Zambon (UK) Ltd., London W6, UK
Abstract
The effect of two fl2-adrenoceptor agonists, salbutamol (100 lag/kg i.v.) and broxaterol (100 lag/kg i.v.), on airway microvascular leakage induced by vagal stimulation was studied in anaesthetised guinea pigs. Airway microvascular leakage was measured by Evans blue extravasation. Broxaterol, but not salbutamol, inhibited Evans blue dye extravasation at all airway levels, an effect prevented by pretreatment with propranolol (1 mg/kg). Neither of the fl2-agonists had any effect on substance P-induced Evans blue dye extravasation. Broxaterol inhibits the prejunctional release of tachykinins from airway sensory nerves by stimulation of fl-receptors. The mechanism by which fl-adrenoceptor agonists prevent airway microvascular leakage deserves further study.
Introduction
Airway microvascular leakage is an integral feature of airway inflammation that is characteristic of asthmatic airways [1]. It has been suggested that the airway inflammation may be mediated in part by neural mechanisms [2]. Airway neurogenic inflammation can be demonstrated in rodents such as rats and guinea pigs by stimulation of the distal ends of sectioned cervical vagus nerves. Stimulation of these nerves has been shown to lead to the release of neuropeptides [3], and to cause an increase in airway microvascular permeability [4] in guinea pig airways in vivo. fl2-adrenoceptor agonists are long-established drugs in asthma therapy, and are used primarily as bronchodilators to relax airway i Author for correspondence: Dr. K. F. Chung, Dept. of Thoracic Medicine, National Heart & Lung Institute, Dovehouse Street, London, SW3, 6LY.
smooth muscle, fl2-adrenoceptor agonists may also inhibit bronchoconstriction mediated by neural mechanisms, by inhibiting the prejunctional release of acetylcholine from cholinergic nerves in human bronchial tissues in vitro [5]. The effects of flE-adrenoceptor agonists on non-cholinergic neural transmission in vivo have not been studied. We postulated that fl2-adrenergic agonists may also inhibit non-cholinergic neurogenic inflammation. We therefore investigated the effects of two selective flz-adrenoceptor agonists, salbutamol and broxaterol, on airway microvascular leakage induced by vagal stimulation and substance P. Broxaterol is a relatively new isoxazole derivative with fl2-adrenoceptor agonist activity [6] and specifically binds to fl2-adrenoceptors in lung membrane preparations [7]. In the conscious guinea pig, broxaterol was as potent as salbutamol in inhibiting histamine and serotonin-induced bronchospasm.
30 Methods
Male Dunkin-Hartley guinea piga (300-350g) were anaesthetised with an intraperitoneal injection of urethane (0.2 g/kg; Sigma, UK) and further doses were given as needed. A jugular vein was cannulated for intravenous drug administration. The cervical vagus nerves were carefully dissected and sectioned. The caudal ends of the cut vagus nerves were then placed on platinum electrodes. Airway microvascular permeability was measured by a modification of the method used by Saria and Lundberg [8], using Evans blue dye as a serum albumin marker. The extravasation of Evans blue dye into airway tissue would reflect plasma protein exudation due to changes in airways' microvascular permeability. This has been validated in our laboratory and extravasation of Evans blue dye was found to correlate well with extravasation of radiolabelled albumin [-9]. Evans blue dye (30 mg/ml) was slowly injected intravenously, 30 s prior to stimulation of both vagus nerves at 5 Hz, 5 mV, and 5 ms for 3 min. Five minutes after vagal stimulation, the animal was perfused with 0.9% NaC1 through a left ventriculotomy to wash off intravascular Evans blue dye. The airways and lungs were then removed en bloc and cleared of extraneous connective tissue and lung parenchyma. The airway was then separated into the trachea, main bronchi and intrapulmonary airways. Each tissue specimen was then blotted dry with filter paper and weighed. Evans blue dye was then extracted by overnight incubation in formamide (2 ml) at 40 ~ in a water bath. The amount of Evans blue dye was quantified by measuring absorbance at 620 nm with a spectrophotometer (Model PU8630, Phillips, Cambridge, UK) and interpolation on a standard curve of absorbance of Evans blue dye with concentrations from 0.25 to 10 ~tg/ml. The amount of Evans blue dye extravasated into the tissue was then expressed as ng/mg of wet weight tissue.
Agents Actions36 (1992) because in previous studies the EDso for intravenous doses of the fl2-adrenoceptor agonists in reversing induced bronchoconstriction was found to be approximately 50 gg/kg for broxaterol and salbutamol (Zambon Co., Milan, Italy; data on file and shown in Table 1). In addition, a group of four guinea pigs had their vagus nerves dissected and placed on platinum electrodes, but not stimulated and pretreated, and served as the sham-stimulated group. In order to determine whether the inhibitory effect on airway microvascular leakage observed with broxaterol was mediated through fl2-adrenergic receptors, further experiments in two groups of six animals (treated with either broxaterol or control) were carried out in the presence of propranolol (1 mg/kg intravenously). All animals were pretreated with intravenous atropine (1 mg/kg) 1 rain before the beginning of vagal stimulation. Another series of experiments was carried out to investigate the effect of fl~-adrenoceptor agonists on substance P-induced airway microvascular leakage which was measured as outlined above. Three groups of four guinea pigs were treated with either control (0.9% saline) or broxaterol (100gg/kg) or salbutamol (100 ~tg/kg), followed 4 min later by atropine and 5 min later by intravenous substance P (1 ng/kg). Statistics
The results are presented as means with standard errors of means. The Kruskal-Wallis analysis of variance was used to detect any significant difference among groups. The M a n n - W h i t n e y U test Table 1
Bronchodilating activity of broxaterol and salbutamol on histamine- and methacholine-induced bronchoconstriction in anaesthetised guinea pigs. EDso values after intravenous administration. Compound
Protocol
Broxaterol
The effect of fl2-adrenoceptor agonists on airway microvascular leakage induced by vagal stimulation was studied in three groups of six animals treated with either intravenous 0.9 % saline control or salbutamol sulphate (100 gg/kg; M W = 239) or broxaterol chloride (100 gg/kg; M W = 263) 10 rain before vagal stimulation. We used these doses
Salbutamol
EDs0 (lag/kgi.v.)" Histamineb
Methacholine b
53 (37-74)* 49 (35-69)
43 (31-61) 49 (33 74)
* Indicates 95% confidencelimits. a ED5o is the dose of broxaterol or salbutamol needed to cause 50% inhibition of the increase in intratracheal pressure induced by histamine or methacholine. b Histamine was given at 20 gg/kg and methacholine at 4 gg/kg.
Agents Actions36 (1992)
31
Table 2
Effect of salinecontrol,salbutamoland broxaterolon Evansblue dye extravasation(gg/mgwet weight)inducedby vagalstimulationin trachea (T), main bronchi (MB) and intrapulmonaryairways (IPA). Saline control
Salbutamol (100gg/kg)
T
125 28 107 35 97 65 Mean 76 SEM 18
Broxaterol (100 gg/kg)
MB
IPA
T
MB
IPA
T
MB
IPA
162 57 163 118 173 96 128 20
117 44 170 34 112 47 87 24
19 47 83 91 93 136 78 18
78 110 174 124 152 215 142 22
19 95 109 80 156 117 96 20
34 34 9 20 16 32 24 5
58 44 32 66 32 44 46 6
23 58 31 25 20 34 32 6
Each set of figures is from the individualguinea pig. Six guinea pigs in each group.
was used to confirm significant differences between any two groups.
not affect substance P-induced Evans blue dye extravasation (Table 4).
Results Discussion
In the saline-treated group, non-cholinergic vagal stimulation caused extensive Evans blue dye extravasation at all airway levels compared to sham stimulation (Table 2). Broxaterol significantly inhibited Evans blue dye extravasation (p
0065-4299/92/020029-04 $1.50 + 0.20/0 9 1992 Birkhfiuser Verlag, Basel
Modulation of neurally mediated airway microvascular leakage in guinea-pig airways by fl2-adrenoceptor agonists K. P. Hui, P. Ventresca, A. C. Brown 1, p. j. Barnes and K. F. Chung Department of Thoracic Medicine, National Heart & Lung Institute, Royal Brompton National Heart & Lung Hospital, London, SW3, UK and i Zambon (UK) Ltd., London W6, UK
Abstract
The effect of two fl2-adrenoceptor agonists, salbutamol (100 lag/kg i.v.) and broxaterol (100 lag/kg i.v.), on airway microvascular leakage induced by vagal stimulation was studied in anaesthetised guinea pigs. Airway microvascular leakage was measured by Evans blue extravasation. Broxaterol, but not salbutamol, inhibited Evans blue dye extravasation at all airway levels, an effect prevented by pretreatment with propranolol (1 mg/kg). Neither of the fl2-agonists had any effect on substance P-induced Evans blue dye extravasation. Broxaterol inhibits the prejunctional release of tachykinins from airway sensory nerves by stimulation of fl-receptors. The mechanism by which fl-adrenoceptor agonists prevent airway microvascular leakage deserves further study.
Introduction
Airway microvascular leakage is an integral feature of airway inflammation that is characteristic of asthmatic airways [1]. It has been suggested that the airway inflammation may be mediated in part by neural mechanisms [2]. Airway neurogenic inflammation can be demonstrated in rodents such as rats and guinea pigs by stimulation of the distal ends of sectioned cervical vagus nerves. Stimulation of these nerves has been shown to lead to the release of neuropeptides [3], and to cause an increase in airway microvascular permeability [4] in guinea pig airways in vivo. fl2-adrenoceptor agonists are long-established drugs in asthma therapy, and are used primarily as bronchodilators to relax airway i Author for correspondence: Dr. K. F. Chung, Dept. of Thoracic Medicine, National Heart & Lung Institute, Dovehouse Street, London, SW3, 6LY.
smooth muscle, fl2-adrenoceptor agonists may also inhibit bronchoconstriction mediated by neural mechanisms, by inhibiting the prejunctional release of acetylcholine from cholinergic nerves in human bronchial tissues in vitro [5]. The effects of flE-adrenoceptor agonists on non-cholinergic neural transmission in vivo have not been studied. We postulated that fl2-adrenergic agonists may also inhibit non-cholinergic neurogenic inflammation. We therefore investigated the effects of two selective flz-adrenoceptor agonists, salbutamol and broxaterol, on airway microvascular leakage induced by vagal stimulation and substance P. Broxaterol is a relatively new isoxazole derivative with fl2-adrenoceptor agonist activity [6] and specifically binds to fl2-adrenoceptors in lung membrane preparations [7]. In the conscious guinea pig, broxaterol was as potent as salbutamol in inhibiting histamine and serotonin-induced bronchospasm.
30 Methods
Male Dunkin-Hartley guinea piga (300-350g) were anaesthetised with an intraperitoneal injection of urethane (0.2 g/kg; Sigma, UK) and further doses were given as needed. A jugular vein was cannulated for intravenous drug administration. The cervical vagus nerves were carefully dissected and sectioned. The caudal ends of the cut vagus nerves were then placed on platinum electrodes. Airway microvascular permeability was measured by a modification of the method used by Saria and Lundberg [8], using Evans blue dye as a serum albumin marker. The extravasation of Evans blue dye into airway tissue would reflect plasma protein exudation due to changes in airways' microvascular permeability. This has been validated in our laboratory and extravasation of Evans blue dye was found to correlate well with extravasation of radiolabelled albumin [-9]. Evans blue dye (30 mg/ml) was slowly injected intravenously, 30 s prior to stimulation of both vagus nerves at 5 Hz, 5 mV, and 5 ms for 3 min. Five minutes after vagal stimulation, the animal was perfused with 0.9% NaC1 through a left ventriculotomy to wash off intravascular Evans blue dye. The airways and lungs were then removed en bloc and cleared of extraneous connective tissue and lung parenchyma. The airway was then separated into the trachea, main bronchi and intrapulmonary airways. Each tissue specimen was then blotted dry with filter paper and weighed. Evans blue dye was then extracted by overnight incubation in formamide (2 ml) at 40 ~ in a water bath. The amount of Evans blue dye was quantified by measuring absorbance at 620 nm with a spectrophotometer (Model PU8630, Phillips, Cambridge, UK) and interpolation on a standard curve of absorbance of Evans blue dye with concentrations from 0.25 to 10 ~tg/ml. The amount of Evans blue dye extravasated into the tissue was then expressed as ng/mg of wet weight tissue.
Agents Actions36 (1992) because in previous studies the EDso for intravenous doses of the fl2-adrenoceptor agonists in reversing induced bronchoconstriction was found to be approximately 50 gg/kg for broxaterol and salbutamol (Zambon Co., Milan, Italy; data on file and shown in Table 1). In addition, a group of four guinea pigs had their vagus nerves dissected and placed on platinum electrodes, but not stimulated and pretreated, and served as the sham-stimulated group. In order to determine whether the inhibitory effect on airway microvascular leakage observed with broxaterol was mediated through fl2-adrenergic receptors, further experiments in two groups of six animals (treated with either broxaterol or control) were carried out in the presence of propranolol (1 mg/kg intravenously). All animals were pretreated with intravenous atropine (1 mg/kg) 1 rain before the beginning of vagal stimulation. Another series of experiments was carried out to investigate the effect of fl~-adrenoceptor agonists on substance P-induced airway microvascular leakage which was measured as outlined above. Three groups of four guinea pigs were treated with either control (0.9% saline) or broxaterol (100gg/kg) or salbutamol (100 ~tg/kg), followed 4 min later by atropine and 5 min later by intravenous substance P (1 ng/kg). Statistics
The results are presented as means with standard errors of means. The Kruskal-Wallis analysis of variance was used to detect any significant difference among groups. The M a n n - W h i t n e y U test Table 1
Bronchodilating activity of broxaterol and salbutamol on histamine- and methacholine-induced bronchoconstriction in anaesthetised guinea pigs. EDso values after intravenous administration. Compound
Protocol
Broxaterol
The effect of fl2-adrenoceptor agonists on airway microvascular leakage induced by vagal stimulation was studied in three groups of six animals treated with either intravenous 0.9 % saline control or salbutamol sulphate (100 gg/kg; M W = 239) or broxaterol chloride (100 gg/kg; M W = 263) 10 rain before vagal stimulation. We used these doses
Salbutamol
EDs0 (lag/kgi.v.)" Histamineb
Methacholine b
53 (37-74)* 49 (35-69)
43 (31-61) 49 (33 74)
* Indicates 95% confidencelimits. a ED5o is the dose of broxaterol or salbutamol needed to cause 50% inhibition of the increase in intratracheal pressure induced by histamine or methacholine. b Histamine was given at 20 gg/kg and methacholine at 4 gg/kg.
Agents Actions36 (1992)
31
Table 2
Effect of salinecontrol,salbutamoland broxaterolon Evansblue dye extravasation(gg/mgwet weight)inducedby vagalstimulationin trachea (T), main bronchi (MB) and intrapulmonaryairways (IPA). Saline control
Salbutamol (100gg/kg)
T
125 28 107 35 97 65 Mean 76 SEM 18
Broxaterol (100 gg/kg)
MB
IPA
T
MB
IPA
T
MB
IPA
162 57 163 118 173 96 128 20
117 44 170 34 112 47 87 24
19 47 83 91 93 136 78 18
78 110 174 124 152 215 142 22
19 95 109 80 156 117 96 20
34 34 9 20 16 32 24 5
58 44 32 66 32 44 46 6
23 58 31 25 20 34 32 6
Each set of figures is from the individualguinea pig. Six guinea pigs in each group.
was used to confirm significant differences between any two groups.
not affect substance P-induced Evans blue dye extravasation (Table 4).
Results Discussion
In the saline-treated group, non-cholinergic vagal stimulation caused extensive Evans blue dye extravasation at all airway levels compared to sham stimulation (Table 2). Broxaterol significantly inhibited Evans blue dye extravasation (p
