J. Auton. Phannacol. (1992). 12, 403-409

Changes in airway reactivity to exogenous and endogenous acetylcholine and substance P after anaphylactic bronchoconstriction in anaesthetized guinea-pigs Paola Nieri, Luisa Daffonchio", Claudio Ominit, Enrica Martinotti & Maria C. Breschi Institute of Biological Sciences, Faculty of Pharmacy, Via Bonanno n. 6, 56100 Pisa, Italy. *Institute ofPharmacologica1 Sciences, Faculty of Pharmacy, Via Balzaretti n.9, 20133 Milan, Italy and fDompe' Farmaceutici S.p.A., Via S. Martino n. 12, 20122 Milan,Italy

1 In anaesthetized, actively sensitized guinea-pigs, the anaphylactic shock induced by antigen aerosol challenge (5 s; 50 mg ml-l) was followed by increase in airway reactivity to both acetylcholine and substance P. In particular dose-response curves to acetylcholine (3-1000 pg kg-' i.v.) and to substance P (5-80 pg kg-' 1.v.) obtained in antigen exposed animals were significantly shifted to the left of those performed in control guinea-pigs (exposed to saline aerosol). 2 The hyperreactive phenomenon after antigen aerosol was also evident when capsaicin-induced bronchoconstriction ( 1-4 pg kg-I i.v.) was tested; the degree of hyperresponsiveness was similar to that observed with acetylcholine and substance P as agonists.

3 The frequency-response curves to vagal stimulation, either cholinergic or NANC in nature, were not significantly modified in guinea-pigs challenged with the antigen in respect to those aerosolized with saline. 4 The data obtained in the present study indicate that the airway hyperresponsi-

veness present in the animal model used is non-specific, involving both cholinergic and peptidergic effects. On the other hand, the lack of potentiation of the bronchoconstriction response to electrical stimulation might suggest that the establishment of a clear hyperreactive phenomenon is under the control of different mechanisms unrelated to increased bronchial reactivity.

Correspondence: Dr Paola Nieri, Institute of Biological Sciences, Faculty of Pharmacy, Via Bonanno n.6, 56100 Pisa, Italy.

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Introduction

Many studies regarding asthma focused their attention on airway hyperreactivity since this phenomenon is a main hallmark of this pathology; in fact, enhanced airway responsiveness to different chemical and pharmacologic stimuli has been widely recognized in asthmatic patients (Boushey, Holtzman, Sheller & Nadel, 1980). The molecular basis of the hyperresponsiveness is not clearly defined; however, inflammation has been indicated as a common underlying alteration of airway hyperreactivity (Barnes, Chung & Page, 1988). The involvement of neural pathways has also been considered and a possible role of the autonomic nervous system in excessive irritability of the tracheobronchial tree in asthmatics has been suggested (Reed, 1974; Barnes, 1986a; Remanske & Kaliner, 1990). Since the parasympathetic nerves are the dominant neural bronchoconstrictor mechanism in most species, an overactivity of this system and an exaggeration of vagal reflex mechanisms have been proposed as a possible explanation of airway hyperreactivity (Gold, 1975; Widdicombe, 1979). More recently it has been suggested that also axon reflexes via the NANC excitatory fibres, leading to neurogenic inflammation, could play a relevant role in asthma (Barnes, 1986b, 1987). As previously described, the 'immediate' anaphylactic response triggered by aerosol antigen challenge in active sensitized guineapig has been demonstrated to result in airway hyperreactivity (Daffonchio, Payne, Lees & Whittle, 1988, 1989), as shown by an increase in serotonin- and acetylcholine-induced bronchoconstriction. The aim of the present study was to verify if the hyperreactive phenomenon could be extended also to substance P, a representative neuropeptide involved in neurogenic inflammation (Saria & Lundberg 1985). Moreover, in an attempt to reveal possible alterations in the activation of neuronal pathways (cholinergic or peptidergic), we have compared the responses obtained with exogenously administered agonists (acetylcholine and substance P) to those induced by the same putative mediators

endogenously released by electrically induced vagal stimulation. In some experiments, the endogenous release of sensory neuropeptides was also induced pharmacologically, by administering capsaicin, the pungent ingredient in peppers of the Capsicum family which is able to stimulate C-afferent fibres (Lundberg & Saria, 1982).

Materials and methods

Active sensitization Male Dunkin-Hartley guinea-pigs (400-500 g; Rodentia, Italy), were actively sensitized in the following way: 17-25 days before the experiments two injections of ovalbumin (OA; Sigma grade 111; 100 mg kg-') were made in the same day, one i.p. and the other S.C.

Anaphylactic challenge The animals were anaesthetized with pentothal sodium (50-70 mg kg-I i.p.) and then the trachea and a jugular vein were cannulated. Vagal nerves were cut in all animals according to Daffonchio et al. (1988). The tracheal cannula was connected to a rodent ventilator pump (Basile Mod. 7025) with the following parameters: 50 strokes min-' of 1 ml of room air per 100 g of animal weight. Pulmonary inflation pressure (PIP, mmHg) was measured by a Bentley-Trantec pressure transducer (Basile Mod. 800) connected to the inspiration line of the ventilator circuit and recorded by means of a Unirecord Microdinamometer (Basile Mod. 7050). The anaphylactic shock was induced with an aerosol of 5 s duration generated from an OA saline solution at the concentration of 50 mg ml-' by a Pulmosonic nebulizer (Devilbiss). Sensitized animals, used as controls, received an aerosol of saline for 5 s. To verify the occurrence of sensitization in these guinea-pigs, at the end of the experiments, OA (5 mg kg-I 1.v.) was injected; the animals which did not respond to antigen with a sustained bronchoconstriction were eliminated from the study.

A I R W A Y HYPERREACTIVITY AFTER A N A P H Y L A C T I C CHALLENGE

Functional studies Dose-response curves for the bronchoconstrictor effect of acetylcholine (ACh; 3- 1000 pg kg-' i.v.), substance P (SP; 5-80 pg kg-' i.v.) and capsaicin 1-4 (Jig kg-' iv.), and frequency-response curves to vagal stimulation (see below) were obtained 60 min after antigen or saline aerosol as indicated by Daffonchio et al. (1 988). The endogenous release of acetylcholine and sensory neuropeptides from the airway vagal fibres was obtained with stimulation of the cervical portion of the vagi. The vagal nerves were placed on a Dastre electrode (Harvard Apparatus) and covered with liquid paraffin 45 rnin after the aerosol in order to avoid a more prolonged stress of the nerves. The electrode was connected to an electrical stimulator (Biomedica Mangoni Mod. Digit 3-T). Acetylcholine release was obtained with the following stimulus parameters: 20 V; 0.3 ms pulse width; 2-32 Hz; 3 s train duration; 8 rnin interval between two trains. The possible involvement of peptides in the bronchoconstriction triggered by these stimulation parameters was excluded from the disappearance of the response with an infusion of atropine (100 pg kg-' min-I i.v.) in separate experiments ( n= 10). Simultaneous release of acetylcholine and neuropeptides was obtained according to Lundberg, Saria, Brodin, Rose11 & Folkers (1 983), with the following stimulus parameters: 20 V; 5 ms pulse width; 0.5-16 Hz; 30 s train duration; 8 rnin interval between two trains. The cholinergic component of the response was removed by atropine infusion (100 pg kg-' min-' i.v.), which started 10 min before the frequency-response curve. Drugs The following drugs were used: acetylcholine hydrochloride, substance P (acetate salt), capsaicin, atropine sulphate, ovalbumin (Grade 111) from Sigma (St Louis, MO, USA) and pentothal sodium from Abbott (Campoverde, LT, Italy). All drugs were dissolved in saline solution, before use, except capsaicin which was dissolved as follows: 10% ethanol, 10% tween 80 and the remaining volume of saline.

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Statistical analysis Data were calculated as increase in PIP over the basal values and expressed as mean? SEM from n replications. Comparisons between dose or frequency-response curves in the different experimental groups were made by means of a computer-aided program based on the variance analysis for parallel regression lines (Snedecor & Cochran, 1967), which allows calculation of the dose-ratio and its 95% confidence limits taking into account all the dose-response curves for each of the two groups under evaluation, according to Finney (1978). Results

An aerosol of ovalbumin (50 mg m1-l; 5 s) induced a maximal anaphylactic bronchoconstriction as shown by a mean PIP increment of 43.99k2.49 mmHg (n=43). A representative trace of this effect and its time-course is reported in Fig. 1. As can be seen, the bronchoconstriction returned progressively to basal values even if a minimal residual increase in PIP (3.86k 0.50 mmHg, n=43) was still present 60 rnin after OA aerosol.

t Oh

aerosol

Fig. 1. A representative trace showing anaphylactic bronchospasm after an ovalbumin (OA) aerosol (50 mg ml-I; 5 s) in anaesthetized guinea-pigs. The peak in bronchoconstriction is followed by a progressive but not complete return to the basal PIP.

The anaphylactic shock was followed by the onset of airway hyperreactivity, as shown by the potentiated bronchoconstrictor response to both exogenously administered acetylcholine and substance P (Fig. 2; Table 11.

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Table 1. Dose-ratio (DR) values and relative 95% confidence limits calculated between doseand frcquency-response curves obtained in control and antigen challenged guinea-pigs 9 5 '/o confidence limits

Bronchoconstrictor challenge

DR

Acetylcholine

2.02**

1.29-3.16

Vagal stimulation (cholinergic response) Substance P

1.15

0.79-1.68 1.24-2.59

Vagal stimulation (NANC response) Capsaicin

1.19**

A-A A

-A

controls n = 8 challenged with OA n = 9

1 m

40 - -

1

E E B a

20 --

I

0-

1.23 1.66**

0.57-2.19 1.1 1-2.50

Ach pg kg-' i.v.

A-A

**Pt0.01

A

80

-A

controls n = 6 challenged with OA n = 6 T

T

Dose-response curves to acetylcholine 60.(3-1000 pg kg-' i.v.), obtained 60 min after I" E E antigen aerosol (n=9), were significantly 40(PtO.0 1) shifted to the left of controls (n= 8) with a DR of 2.02 (95% confidence limits: 20 -1.29-3.16). Similar results were obtained for the bronchoconstriction induced by subI O U stance P and indeed the DR calculated 10 20 40 80 5 between dose-response curves (5-80 p g kg-' SP pg kg-' i.v. i.v.) performed in control or antigen exposed (n=6 for each group) guinea-pigs was 1.79 Fig. 2. Dose-response curves to Ach (top) and (95% confidence limits: 1.24-2.59; P

Changes in airway reactivity to exogenous and endogenous acetylcholine and substance P after anaphylactic bronchoconstriction in anaesthetized guinea-pigs.

1. In anaesthetized, actively sensitized guinea-pigs, the anaphylactic shock induced by antigen aerosol challenge (5 s; 50 mg ml-1) was followed by in...
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