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Inhibitory effects of clonidine on bronchospasm induced in guinea-pigs by vagal stimulation or antigen challenge
European Journal of Pharmacology, 123 (1986) 181-185
181
Elsevier
I N H I B I T O R Y EFFECTS OF C L O N I D I N E O N B R O N C H O S P A S M BY VAGAL S T I M U L A T I O N O R A N T I G E N C H A L L E N G E
I N D U C E D IN G U I N E A - P I G S
R.G.G. ANDERSSON *, A. FOGNER, B.R. LINDGREN and G. MUACEVIC
Department of Pharmacology, University of LinkOping, S-58185 Link6ping, Sweden, and Department of Pharmacology, Boehringer Ingelheim KG, D-6507 lngelheim /Rhein, F.R.G. Received 14 November 1985, accepted 21 January 1986
R.G.G. ANDERSSON, A. FUGNER, B.R. LINDGREN and G. MUACEVIC, Inhibitory effects of clonidine on bronchospasm induced in guinea-pigs by vagal stimulation or antigen challenge, European J. Pharmacol. 123 (1986) 181-185. The effects of clonidine on the bronchospastic responses induced by vagal stimulation or antigen challenge were studied in anaesthetized guinea-pigs. Electrical stimulation of the vagus nerves by 2-4 Hz induced a vigorous, mainly atropine-sensitive bronchoconstriction, which was strongly inhibited by clonidine (0.05 mg/kg i.v.). The inhibitory effect of clonidine was significantly reduced by the a2-adrenoceptor antagonist yohimbine (1 mg/kg i.v.). Another series of experiments was done with ovalbumin-sensitized guinea-pigs. Respiratory anaphylaxis was induced by antigen inhalation resulting in an increase of pulmonary resistance from 100% (baseline) to about 190% in the control group. Animals pretreated with a clonidine aerosol (0.03%) showed a marked inhibition of the bronchospastic response. It is suggested that the inhibition of the bronchospastic responses induced by clonidine may be mediated by a stimulation of a2-adrenoceptors, which exerts an inhibitory control of the excitatory vagal activity in the guinea-pig airways. Bronchospasm
Clonidine
Inhibitory effects
1. Introduction Catecholamines have for a long time proved to be useful remedies in the treatment of bronchoobstructive diseases (see Svedmyr and Simonsson, 1978). Traditionally, their effects have been ascribed to the relaxation of the airway smooth muscles by stimulation of/3-adrenoceptors. However, we have recently shown that dilatation of trachea and bronchi of the guinea-pig may also be induced by stimulation of az-adrenoce ptors located prejunctionally on cholinergic neurones. When these receptors were stimulated, smooth muscle relaxation was induced secondary to the inhibition of cholinergic neurotransmission * To whom all correspondence should be addressed: Department of Pharmacology, University of Link~Sping, S-58185 LinkiSping, Sweden. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
(Grundstr~Sm et al., 1981a,b). Both clonidine and B - H T 920 were potent inhibitors of cholinergic neurotransmission in guinea-pig airway (Wikberg et al., 1982). Clonidine was also found to inhibit the antigen-induced, IgE-mediated release of histamine from human leucocytes. This effect was suggested to occur through stimulation of H2-receptors, as H2-antihistamines antagonized this effect (Andersson et al., 1978). Comparatively little research has been done on the bronchopulmonary effects of a2-adrenoceptor agonists in vivo, although these receptors are known to play an important role in other physiological functions, e.g. blood pressure regulation (Schmitt, 1977). Recently, however, Advenier et al. (1983) demonstrated that clonidine enhanced the bronchoconstrictor effects of histamine, acetylcholine and serotonin in the guinea-pig. GrundstrOm and Andersson (1985), however, showed
182 that clonidine inhibited the increase in respiratory insufflation pressure caused by stimulation of non-cholinergic excitatory nerves in the guinea-pig. In the present work, we have studied the effects of clonidine on the bronchoconstriction induced by electrical stimulation of the vagus nerve in guinea-pigs anaesthetized with urethane. Ovalbumin-sensitized guinea-pigs were used in some experiments and bronchoconstriction was induced by ovalbumin challenge.
2. Materials and methods 2.1. Animals
Male albino guinea-pigs (Pirbright-White) of 370-440 g body weight were used in the vagus stimulation tests. Guinea-pigs (mixed breed) of 250-300 g body weight were used for the sensitization. They were sensitized by one i.p. injection of 10 /~g of ovalbumin together with 100 mg of AI(OH)3 adjuvant and used 14 days later. 2.2. Experiments on non-sensitized animals
The guinea-pigs were anaesthetized with urethane (1.8 g / k g i.p.) and provided with a tracheal cannula. A catheter was inserted into the left jugular vein and the animals were given gallamine (3 m g / k g i.v.), placed in a supine position in a body plethysmograph and connected to a respiratory (Rhema) at a rate of 60 strokes/min. Ventilation pressure was kept constant (10 cm water column) by a water valve (via a sidearm of the cannula) providing a tidal volume of 2.5-3 ml. The changes of pressure in the body box during respiration were recorded through a Statham (PM5 ETC) recorder on a Grass polygraph as described by Gjuris et al. (1964). The body temperature of the animal was maintained at 38°C, using a heater control device. All guinea-pigs had both cervical vagal nerves divided at the beginning of the experiments; the distal ends were stimulated with electrodes at 2-4 V per ms, 2-5 Hz for 20 s using a Grass $4 stimulator. Using the same method for evaluation, other guinea-pigs were not stimulated electrically but with histamine (0.01-0.02 m g / k g
i.v.) to assess the effects of clonidine and propranolol. Clonidine was pretested i.v. (0.025, 0.05, 0.1 and 0.15 mg/kg) for inhibition of bronchospasm due to electrical stimulation so as to select a near EDs0 dose (0.05 mg/kg) for administration in the main experiments. 2.3. Experiments on sensitized animals
The guinea-pigs were anaesthetized with urethane, 1.4 g / k g i.p., provided with a tracheal cannula and a water-filled esophageal catheter and were placed in a supine position into a whole-body plethysmograph. Half an hour was allowed for adaptation of the spontaneously breathing animals while the normal lung function was checked intermittently. The esophageal pressure, equivalent to pleural pressure, was measured by connecting one inlet of a differential pressure transducer (Validyne MP 45 + 20 cm H 2 0 ) to the esophageal catheter with the other inlet open to the atmosphere. Esophageal pressure was thus measured in place of transpulmonary pressure. The air flow rate was determined via a Fleisch pneumotachograph connected to a Validyne (MP 45 + 2 cm H 2 0 ) transducer and fitted to the rear end of the plethysmograph (cf. diagram in Fiagner, 1985). Both flow and pressure signals were fed into an on-line analog computer (Buxco Electronics Inc., Sharon, CT) which analyses pulmonary mechanics on a breath-by-breath basis. All parameters were digitalized by a data logger (Model DL-12, Buxco) and recorded on a data terminal (Texas Instruments, Silent 700). Each quinea-pig was challenged by inhalation of antigen aerosol (1% solution of ovalbumin) for a 60 s period (minimal volume + S.E.: 125 + 4.6 ml) using an ultrasonic nebulizer (particle size 1-6 /~m). Increase of pulmonary resistance (R-L) as a result of antigen exposure was compared in drugtreated and vehicle-treated animals and differences were expressed as % inhibition. 2.4. Drugs
The following drugs were used: atropine sulphate and clonidine hydrochloride (Boeh-
183
ringer-Ingelheim, Ingelheim, FRG), prazosin hydrochloride (Pfizer Inc., CT, USA), yohimbine hydrochloride (Sigma Chemical Co., St. Louis, MO, USA).
IB
ClC
-
I 2 Hl
T 2Hz
L
+J
I
t
2.5. Statistical evaluation
The results of table 1 were evaluated by means of the analysis of variance for block designs, with the animals as blocks. Group comparisons were carried out by using orthogonal contrasts. In the experiment with sensitized animals (fig. 2A) the t-tests were carried out for each individual time separately and should be regarded as explorative.
3. Results
Electrical stimulation of the vagal nerves caused a marked bronchoconstriction in anaesthetized guinea-pigs. While 2 Hz induced a partial change, 3-4 Hz induced a vigorous bronchospasm (fig. 1A-C). The bronchoconstriction caused by vagus stimulation was mainly inhibited by atropine (0.01 mg/kg) (fig. 1D). Clonidine (0.05 mg/kg) administered i.v. to anaesthetized animals did not change the basal airway resistance but significantly (P < 0.01) inhibited the bronchospasm induced by stimulation of vagal nerves (table 1, fig. 1E). An aE-adrenoceptor antagonist, yohimbine (1 mg/kg), reduced the inhibitory effect of clonidine while the al-adrenoceptor antagonist prazosin (0.03 mg/kg) TABLE 1 Effect of clonidine (0.05 mg/kg i.v.) on bronchospasm induced by electrical stimulation of the vagus nerve. In some tests the animals were pretreated with yohimbine (1 mg/kg i.v.) or prazosin (0.03 mg/kg i.v.). Mean values for bronchospasm+ S.E.M.; n = 5. The mean values for clonidine and prazosin+ clonidine differ significantly (P < 0.01) from that for yohimbine + clonidine as well as from the control value. No significant difference was seen between clonidine and prazosin treatments. Treatment
Control Clonidine Yohimbine Prazosin +clonidine +clonidine
Bronchospasm (%)mean 53 S.E.M. 5
22 4
44 8
15 4
v
I+
Fig. 1. Effects of drugs on responses induced by vagal stimulation of guinea-pig airways. (A-C) stimulation from 2 to 4 Hz; (D) effect of atropine (0.01 mg/kg i.v.); (E) inhibitory influence of clonidine (0.05 mg/kg i.v.); (F and G) effect of clonidine in the presence of prazosin (0.03 mg/kg i.v.) and yohimbine (1 mg/kg i.v.), respectively.
did not prevent the effect of clonidine (table 1, fig. 1). In some experiments, a bronchospasm was induced by histamine (0.01-0.02 mg/kg). Clonidine (0.05 mg/kg i.v.) as well as propranolol (0.2 mg/kg i.v.) increased the bronchospasm induced by histamine by 15.5 + 3.5 and 67 + 13% (~ + S.E.M.), respectively. In combination with propranolol, clonidine did not further (1.7 + 3.7%) increase the bronchospasm induced by histamine. Sensitized guinea-pigs were used in a further series of experiments. Respiratory anaphylaxis was induced in the guinea-pigs by antigen (ovalbumin) inhalation resulting in an increase of pulmonary resistance from 100 (baseline) to about 190% in the control group. Animals pretreated with a clonidine aerosol (0.03%) showed a marked inhibition of this bronchospastic response (fig. 2A). Inhibition was most pronounced between 1.8 and 5 min (P < 0.05) after challenge. Increasing the concentration of the clonidine aerosol to 0.1% did not lead to further amelioration, and nebulization of a 0.01% solution was ineffective (data not shown).
4. D i s c u s s i o n
The present results give support to the theory that a2-adrenoceptors stimulated by clonidine
184
200-
H
140"
~k
control
clonidinn
// ~_
AG-chollon
~:
"
\,
',
.~ 120100-
rain Fig. 2. Effects of antigen challenge (aerosolized ovalbumin) on pulmonary resistance; attenuation by administration of nebulized clonidine (0.03%) for 30 s. Control group n = 5, clonidine group n = 7.
mediate an inhibitory control of the excitatory vagal activity in the airways of the guinea-pig in vivo. This is in accordance with the in vitro results reported by Grundstr~Sm et al. (1984) indicating that noradrenaline inhibited cholinergic neurotransmission in guinea-pig airways by acting on prejunctional a2-adrenoceptors. The involvement of tz2-adrenoceptors is supported by the finding that the a2-receptor antagonist yohimbine was a potent antagonist both in vivo and in vitro of the inhibitory effects of clonidine and noradrenaline, in contrast to the a:-receptor antagonist prazosin (table 1). In the present investigation the bronchospasm induced by vagal stimulation was mainly due to release of the cholinergic transmitter acetylcholine, since the spasm was almost completely abolished by atropine. In another study (GrundstriSm and Andersson, 1985) an excitatory non-cholinergic component of the vagus nerve was demonstrated in addition to the cholinergic component. The bronchospasm elicited by stimulation of the non-cholinergic nerve was also inhibited by stimulating the a2-adrenoceptors with clonidine. In an additional series of experiments, however, clonidine enhanced the bronchospasm induced by histamine. This is in accordance with the results observed by Advenier et al. (1983), who demonstrated that clonidine enhanced the bronchoconstrictor effects of histamine, acetylcholine and serotonin in conscious guinea-pigs. These authors suggested that clonidine inhibited the sympathetic
activity moderating bronchospasm by specifically stimulating ct2-adrenoceptors. Our results demonstrated an inhibitory influence of catecholamines on the airways exposed to histamine, since the fl-adrenoceptor antagonist, propranolol, markedly enhanced the histamine response. The effect of propranolol was more pronounced than that of clonidine, and when combined with propranolol, clonidine did not further enhance the bronchospasm due to histamine. Mediators released during the immediate hypersensitivity reaction may act directly on bronchi or by stimulating irritant receptors with reflex bronchoconstriction. Extensive studies now exist that indicate that a vagal reflex bronchoconstriction is involved in animals subjected to anaphylactic or allergen-antibody reactions in the lung or to exposure of the airways to chemical mediators thought to be released in these reactions (Widdicombe, 1977). In the present investigation we could demonstrate a clear-cut inhibition of the anaphylactic bronchoconstriction by a clonidine aerosol. Preliminary results with i.v. administered clonidine (0.05 mg/kg; data not shown) also showed inhibition of the antigen-induced bronchoconstriction. This inhibition might have been caused by an inhibitory effect of clonidine on the bronchoconstriction caused by a vagal reflex and/or by inhibition of the allergen-induced histamine release which is of importance during the first phase of the immediate hypersensitivity reaction in
185 guinea-pigs ( C h a n d , 1979). A n i n h i b i t o r y effect of c l o n i d i n e o n the latter m e c h a n i s m has been demonstrated in h u m a n basophil a n d mast cell preparations, where c l o n i d i n e inhibited the antigeni n d u c e d release of histamine ( A n d e r s s o n et al., 1978; L i n d g r e n a n d Andersson, 1983). It was suggested that, in these tissues, clonidine acts via H2-receptors. These receptors are thought to be involved in the feedback i n h i b i t i o n of m e d i a t o r release (Lichtenstein a n d Gillespie, 1975). Some deaths occurred d u r i n g the experiments. T h o u g h u n e x p l a i n e d , a lowering of blood pressure after c l o n i d i n e i n h a l a t i o n might offer a plausible exp l a n a t i o n in the case of these anaesthetized a n d s p o n t a n e o u s l y b r e a t h i n g animals. I n conclusion, this investigation d e m o n s t r a t e d that b r o n c h o s p a s m i n d u c e d in the guinea-pig in vivo b y vagus s t i m u l a t i o n or b y antigen challenge can be inhibited b y clonidine. A part of the effect is p r o b a b l y mediated b y s t i m u l a t i o n of, presumably presynaptic, c~2-adrenoceptors. Recently, we could also d e m o n s t r a t e an i n h i b i t o r y effect of clonidine aerosol o n the b r o n c h o s p a s m i n d u c e d b y antigen challenge in asthmatic patients (Lindgren et al., 1984).
Acknowledgements Mrs. S. Albertsmeier, Mrs. P. Goetze and Miss C. Meissner is greatly appreciated. We thank Dr. F. Knappen for helpful advice on statistical analysis of the data. Supported by grants from the Swedish Medical Research Council (04X-4498; 04P6155), the king Gustaf V 80 Years Foundation, the Swedish National Association Against Chest and Heart Diseases, A.O. Sward's Foundation and Osterg~StlandsL~insLandsting.
References Advenier, C., A. Floch and B. Mallard, 1983, Bronchopulmonary effects of clortidine on the bronchomotor responses of the guinea-pig, European J. Pharmacol. 89, 85.
Andersson, R.G.G., B.R. Lindgren and H. Colldahl, 1978, Inhibitory effects of imidazolines on histamine liberation from human leukocytes and on tracheal smooth muscle tone, Acta Pharmacol. Toxicol. 42, 381. Chand, N., 1979, FPL 55712 - An antagonist of slow reacting substance of anaphylaxis: A review, Agents Actions 9, 133. Fiagner, A., 1985, in: Pulmonary and Antiallergic Drugs, ed. J.P. Devlin (Wiley, New York) p. 123. Gjuris, V., B. Heicke and E. Westermann, 1964, Ober die Stimulierung der Atmung durch Bradykinin and KaUidin, Naunyn-Schmiedeb. Arch. Exp. Path. Pharmakol. 247, 429. GrundstriSm, N. and R.G.G. Andersson, 1985, In vivo demonstration of c~2-adrenoceptor-mediatedinhibition of the excitatory non-cholinergic neurotransmission in guinea-pig airways, Naunyn-Schmiedeb. Arch. Pharmacol. 328, 236. GrundstriSm,N., R.G.G. Andersson and J.E.S. Wikberg, 1981a, Pharmacological characterization of the autonomous innervation of the guinea-pig tracheobronchial smooth muscle, Acta Pharmacol. Toxicol. 49, 150. Grundstr~m, N., R.G.G. Andersson and J.E.S. Wikberg, 1981b, Prejunctional a2-adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission, Life Sci. 28, 2981. GrundstrOm, N., R.R.G. Andersson and J.E.S. Wikberg, 1984, Inhibition of the excitatory non-adrenergic, non-cholinergic neurotransmission in the guinea-pig tracheo-bronchial tree mediated by a2-adrenoceptors, Acta Pharmacol. Toxicol. 54, 8. Lichtenstein, L.M. and E. Gillespie, 1975, The effects of H 1 and H 2 antihistamines on allergic histamine release and its inhibition by histamine, J. Pharmacol. Exp. Ther. 192, 441. Lindgren, B.R. and R.G.G. Andersson, 1983, Effekter av klonidin vid experimentell astma, Hygiea 92, 177. Lindgren, B.R., R.G.G. Andersson and T. Ekstrt~m, 1984, Effekter av klonidin pa antigenprovocerad astma, Hygiea 93, 188. Schmitt, H., 1977, The pharmacology of clonidine and related products, in: Handbook of Experimental Pharmacology (Springer-Verlag, Berlin) pp. 39, 229. Svedmyr, N. and B.G. Simonsson, 1978, Drugs in the treatment of asthma, Pharmacol. Ther. B 3, 397. Widdicombe, J.G., 1977, Reflex control of tracheobronchial smooth muscle in experimental and human asthma, in: Asthma, eds. L.M. Lichtenstein and K.F. Austen (A.P.N.Y.) p. 225. Wikberg, J.E.S., N. Grundstr~3m, P. Visnovsky and R.G.G. Andersson, 1982, Pharmacology of B-HT 920 in some isolated smooth muscle of the guinea-pig, Acta Pharmacol. Toxicol. 50, 266.
181
Elsevier
I N H I B I T O R Y EFFECTS OF C L O N I D I N E O N B R O N C H O S P A S M BY VAGAL S T I M U L A T I O N O R A N T I G E N C H A L L E N G E
I N D U C E D IN G U I N E A - P I G S
R.G.G. ANDERSSON *, A. FOGNER, B.R. LINDGREN and G. MUACEVIC
Department of Pharmacology, University of LinkOping, S-58185 Link6ping, Sweden, and Department of Pharmacology, Boehringer Ingelheim KG, D-6507 lngelheim /Rhein, F.R.G. Received 14 November 1985, accepted 21 January 1986
R.G.G. ANDERSSON, A. FUGNER, B.R. LINDGREN and G. MUACEVIC, Inhibitory effects of clonidine on bronchospasm induced in guinea-pigs by vagal stimulation or antigen challenge, European J. Pharmacol. 123 (1986) 181-185. The effects of clonidine on the bronchospastic responses induced by vagal stimulation or antigen challenge were studied in anaesthetized guinea-pigs. Electrical stimulation of the vagus nerves by 2-4 Hz induced a vigorous, mainly atropine-sensitive bronchoconstriction, which was strongly inhibited by clonidine (0.05 mg/kg i.v.). The inhibitory effect of clonidine was significantly reduced by the a2-adrenoceptor antagonist yohimbine (1 mg/kg i.v.). Another series of experiments was done with ovalbumin-sensitized guinea-pigs. Respiratory anaphylaxis was induced by antigen inhalation resulting in an increase of pulmonary resistance from 100% (baseline) to about 190% in the control group. Animals pretreated with a clonidine aerosol (0.03%) showed a marked inhibition of the bronchospastic response. It is suggested that the inhibition of the bronchospastic responses induced by clonidine may be mediated by a stimulation of a2-adrenoceptors, which exerts an inhibitory control of the excitatory vagal activity in the guinea-pig airways. Bronchospasm
Clonidine
Inhibitory effects
1. Introduction Catecholamines have for a long time proved to be useful remedies in the treatment of bronchoobstructive diseases (see Svedmyr and Simonsson, 1978). Traditionally, their effects have been ascribed to the relaxation of the airway smooth muscles by stimulation of/3-adrenoceptors. However, we have recently shown that dilatation of trachea and bronchi of the guinea-pig may also be induced by stimulation of az-adrenoce ptors located prejunctionally on cholinergic neurones. When these receptors were stimulated, smooth muscle relaxation was induced secondary to the inhibition of cholinergic neurotransmission * To whom all correspondence should be addressed: Department of Pharmacology, University of Link~Sping, S-58185 LinkiSping, Sweden. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
(Grundstr~Sm et al., 1981a,b). Both clonidine and B - H T 920 were potent inhibitors of cholinergic neurotransmission in guinea-pig airway (Wikberg et al., 1982). Clonidine was also found to inhibit the antigen-induced, IgE-mediated release of histamine from human leucocytes. This effect was suggested to occur through stimulation of H2-receptors, as H2-antihistamines antagonized this effect (Andersson et al., 1978). Comparatively little research has been done on the bronchopulmonary effects of a2-adrenoceptor agonists in vivo, although these receptors are known to play an important role in other physiological functions, e.g. blood pressure regulation (Schmitt, 1977). Recently, however, Advenier et al. (1983) demonstrated that clonidine enhanced the bronchoconstrictor effects of histamine, acetylcholine and serotonin in the guinea-pig. GrundstrOm and Andersson (1985), however, showed
182 that clonidine inhibited the increase in respiratory insufflation pressure caused by stimulation of non-cholinergic excitatory nerves in the guinea-pig. In the present work, we have studied the effects of clonidine on the bronchoconstriction induced by electrical stimulation of the vagus nerve in guinea-pigs anaesthetized with urethane. Ovalbumin-sensitized guinea-pigs were used in some experiments and bronchoconstriction was induced by ovalbumin challenge.
2. Materials and methods 2.1. Animals
Male albino guinea-pigs (Pirbright-White) of 370-440 g body weight were used in the vagus stimulation tests. Guinea-pigs (mixed breed) of 250-300 g body weight were used for the sensitization. They were sensitized by one i.p. injection of 10 /~g of ovalbumin together with 100 mg of AI(OH)3 adjuvant and used 14 days later. 2.2. Experiments on non-sensitized animals
The guinea-pigs were anaesthetized with urethane (1.8 g / k g i.p.) and provided with a tracheal cannula. A catheter was inserted into the left jugular vein and the animals were given gallamine (3 m g / k g i.v.), placed in a supine position in a body plethysmograph and connected to a respiratory (Rhema) at a rate of 60 strokes/min. Ventilation pressure was kept constant (10 cm water column) by a water valve (via a sidearm of the cannula) providing a tidal volume of 2.5-3 ml. The changes of pressure in the body box during respiration were recorded through a Statham (PM5 ETC) recorder on a Grass polygraph as described by Gjuris et al. (1964). The body temperature of the animal was maintained at 38°C, using a heater control device. All guinea-pigs had both cervical vagal nerves divided at the beginning of the experiments; the distal ends were stimulated with electrodes at 2-4 V per ms, 2-5 Hz for 20 s using a Grass $4 stimulator. Using the same method for evaluation, other guinea-pigs were not stimulated electrically but with histamine (0.01-0.02 m g / k g
i.v.) to assess the effects of clonidine and propranolol. Clonidine was pretested i.v. (0.025, 0.05, 0.1 and 0.15 mg/kg) for inhibition of bronchospasm due to electrical stimulation so as to select a near EDs0 dose (0.05 mg/kg) for administration in the main experiments. 2.3. Experiments on sensitized animals
The guinea-pigs were anaesthetized with urethane, 1.4 g / k g i.p., provided with a tracheal cannula and a water-filled esophageal catheter and were placed in a supine position into a whole-body plethysmograph. Half an hour was allowed for adaptation of the spontaneously breathing animals while the normal lung function was checked intermittently. The esophageal pressure, equivalent to pleural pressure, was measured by connecting one inlet of a differential pressure transducer (Validyne MP 45 + 20 cm H 2 0 ) to the esophageal catheter with the other inlet open to the atmosphere. Esophageal pressure was thus measured in place of transpulmonary pressure. The air flow rate was determined via a Fleisch pneumotachograph connected to a Validyne (MP 45 + 2 cm H 2 0 ) transducer and fitted to the rear end of the plethysmograph (cf. diagram in Fiagner, 1985). Both flow and pressure signals were fed into an on-line analog computer (Buxco Electronics Inc., Sharon, CT) which analyses pulmonary mechanics on a breath-by-breath basis. All parameters were digitalized by a data logger (Model DL-12, Buxco) and recorded on a data terminal (Texas Instruments, Silent 700). Each quinea-pig was challenged by inhalation of antigen aerosol (1% solution of ovalbumin) for a 60 s period (minimal volume + S.E.: 125 + 4.6 ml) using an ultrasonic nebulizer (particle size 1-6 /~m). Increase of pulmonary resistance (R-L) as a result of antigen exposure was compared in drugtreated and vehicle-treated animals and differences were expressed as % inhibition. 2.4. Drugs
The following drugs were used: atropine sulphate and clonidine hydrochloride (Boeh-
183
ringer-Ingelheim, Ingelheim, FRG), prazosin hydrochloride (Pfizer Inc., CT, USA), yohimbine hydrochloride (Sigma Chemical Co., St. Louis, MO, USA).
IB
ClC
-
I 2 Hl
T 2Hz
L
+J
I
t
2.5. Statistical evaluation
The results of table 1 were evaluated by means of the analysis of variance for block designs, with the animals as blocks. Group comparisons were carried out by using orthogonal contrasts. In the experiment with sensitized animals (fig. 2A) the t-tests were carried out for each individual time separately and should be regarded as explorative.
3. Results
Electrical stimulation of the vagal nerves caused a marked bronchoconstriction in anaesthetized guinea-pigs. While 2 Hz induced a partial change, 3-4 Hz induced a vigorous bronchospasm (fig. 1A-C). The bronchoconstriction caused by vagus stimulation was mainly inhibited by atropine (0.01 mg/kg) (fig. 1D). Clonidine (0.05 mg/kg) administered i.v. to anaesthetized animals did not change the basal airway resistance but significantly (P < 0.01) inhibited the bronchospasm induced by stimulation of vagal nerves (table 1, fig. 1E). An aE-adrenoceptor antagonist, yohimbine (1 mg/kg), reduced the inhibitory effect of clonidine while the al-adrenoceptor antagonist prazosin (0.03 mg/kg) TABLE 1 Effect of clonidine (0.05 mg/kg i.v.) on bronchospasm induced by electrical stimulation of the vagus nerve. In some tests the animals were pretreated with yohimbine (1 mg/kg i.v.) or prazosin (0.03 mg/kg i.v.). Mean values for bronchospasm+ S.E.M.; n = 5. The mean values for clonidine and prazosin+ clonidine differ significantly (P < 0.01) from that for yohimbine + clonidine as well as from the control value. No significant difference was seen between clonidine and prazosin treatments. Treatment
Control Clonidine Yohimbine Prazosin +clonidine +clonidine
Bronchospasm (%)mean 53 S.E.M. 5
22 4
44 8
15 4
v
I+
Fig. 1. Effects of drugs on responses induced by vagal stimulation of guinea-pig airways. (A-C) stimulation from 2 to 4 Hz; (D) effect of atropine (0.01 mg/kg i.v.); (E) inhibitory influence of clonidine (0.05 mg/kg i.v.); (F and G) effect of clonidine in the presence of prazosin (0.03 mg/kg i.v.) and yohimbine (1 mg/kg i.v.), respectively.
did not prevent the effect of clonidine (table 1, fig. 1). In some experiments, a bronchospasm was induced by histamine (0.01-0.02 mg/kg). Clonidine (0.05 mg/kg i.v.) as well as propranolol (0.2 mg/kg i.v.) increased the bronchospasm induced by histamine by 15.5 + 3.5 and 67 + 13% (~ + S.E.M.), respectively. In combination with propranolol, clonidine did not further (1.7 + 3.7%) increase the bronchospasm induced by histamine. Sensitized guinea-pigs were used in a further series of experiments. Respiratory anaphylaxis was induced in the guinea-pigs by antigen (ovalbumin) inhalation resulting in an increase of pulmonary resistance from 100 (baseline) to about 190% in the control group. Animals pretreated with a clonidine aerosol (0.03%) showed a marked inhibition of this bronchospastic response (fig. 2A). Inhibition was most pronounced between 1.8 and 5 min (P < 0.05) after challenge. Increasing the concentration of the clonidine aerosol to 0.1% did not lead to further amelioration, and nebulization of a 0.01% solution was ineffective (data not shown).
4. D i s c u s s i o n
The present results give support to the theory that a2-adrenoceptors stimulated by clonidine
184
200-
H
140"
~k
control
clonidinn
// ~_
AG-chollon
~:
"
\,
',
.~ 120100-
rain Fig. 2. Effects of antigen challenge (aerosolized ovalbumin) on pulmonary resistance; attenuation by administration of nebulized clonidine (0.03%) for 30 s. Control group n = 5, clonidine group n = 7.
mediate an inhibitory control of the excitatory vagal activity in the airways of the guinea-pig in vivo. This is in accordance with the in vitro results reported by Grundstr~Sm et al. (1984) indicating that noradrenaline inhibited cholinergic neurotransmission in guinea-pig airways by acting on prejunctional a2-adrenoceptors. The involvement of tz2-adrenoceptors is supported by the finding that the a2-receptor antagonist yohimbine was a potent antagonist both in vivo and in vitro of the inhibitory effects of clonidine and noradrenaline, in contrast to the a:-receptor antagonist prazosin (table 1). In the present investigation the bronchospasm induced by vagal stimulation was mainly due to release of the cholinergic transmitter acetylcholine, since the spasm was almost completely abolished by atropine. In another study (GrundstriSm and Andersson, 1985) an excitatory non-cholinergic component of the vagus nerve was demonstrated in addition to the cholinergic component. The bronchospasm elicited by stimulation of the non-cholinergic nerve was also inhibited by stimulating the a2-adrenoceptors with clonidine. In an additional series of experiments, however, clonidine enhanced the bronchospasm induced by histamine. This is in accordance with the results observed by Advenier et al. (1983), who demonstrated that clonidine enhanced the bronchoconstrictor effects of histamine, acetylcholine and serotonin in conscious guinea-pigs. These authors suggested that clonidine inhibited the sympathetic
activity moderating bronchospasm by specifically stimulating ct2-adrenoceptors. Our results demonstrated an inhibitory influence of catecholamines on the airways exposed to histamine, since the fl-adrenoceptor antagonist, propranolol, markedly enhanced the histamine response. The effect of propranolol was more pronounced than that of clonidine, and when combined with propranolol, clonidine did not further enhance the bronchospasm due to histamine. Mediators released during the immediate hypersensitivity reaction may act directly on bronchi or by stimulating irritant receptors with reflex bronchoconstriction. Extensive studies now exist that indicate that a vagal reflex bronchoconstriction is involved in animals subjected to anaphylactic or allergen-antibody reactions in the lung or to exposure of the airways to chemical mediators thought to be released in these reactions (Widdicombe, 1977). In the present investigation we could demonstrate a clear-cut inhibition of the anaphylactic bronchoconstriction by a clonidine aerosol. Preliminary results with i.v. administered clonidine (0.05 mg/kg; data not shown) also showed inhibition of the antigen-induced bronchoconstriction. This inhibition might have been caused by an inhibitory effect of clonidine on the bronchoconstriction caused by a vagal reflex and/or by inhibition of the allergen-induced histamine release which is of importance during the first phase of the immediate hypersensitivity reaction in
185 guinea-pigs ( C h a n d , 1979). A n i n h i b i t o r y effect of c l o n i d i n e o n the latter m e c h a n i s m has been demonstrated in h u m a n basophil a n d mast cell preparations, where c l o n i d i n e inhibited the antigeni n d u c e d release of histamine ( A n d e r s s o n et al., 1978; L i n d g r e n a n d Andersson, 1983). It was suggested that, in these tissues, clonidine acts via H2-receptors. These receptors are thought to be involved in the feedback i n h i b i t i o n of m e d i a t o r release (Lichtenstein a n d Gillespie, 1975). Some deaths occurred d u r i n g the experiments. T h o u g h u n e x p l a i n e d , a lowering of blood pressure after c l o n i d i n e i n h a l a t i o n might offer a plausible exp l a n a t i o n in the case of these anaesthetized a n d s p o n t a n e o u s l y b r e a t h i n g animals. I n conclusion, this investigation d e m o n s t r a t e d that b r o n c h o s p a s m i n d u c e d in the guinea-pig in vivo b y vagus s t i m u l a t i o n or b y antigen challenge can be inhibited b y clonidine. A part of the effect is p r o b a b l y mediated b y s t i m u l a t i o n of, presumably presynaptic, c~2-adrenoceptors. Recently, we could also d e m o n s t r a t e an i n h i b i t o r y effect of clonidine aerosol o n the b r o n c h o s p a s m i n d u c e d b y antigen challenge in asthmatic patients (Lindgren et al., 1984).
Acknowledgements Mrs. S. Albertsmeier, Mrs. P. Goetze and Miss C. Meissner is greatly appreciated. We thank Dr. F. Knappen for helpful advice on statistical analysis of the data. Supported by grants from the Swedish Medical Research Council (04X-4498; 04P6155), the king Gustaf V 80 Years Foundation, the Swedish National Association Against Chest and Heart Diseases, A.O. Sward's Foundation and Osterg~StlandsL~insLandsting.
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