Ewopeuc 0

Journal

of Pharmacology,

1991 Elsevier

ADONfS

Science Publishers 001429999100605S

202 (199

B.V.

1)97-99

All rights reserved

0014-2999/91/$03.50

EJP 20901

Short communication

Luisa Daffonchio, Alicia Hernandez and Claudio Omini * Institute of Pharmacologicd

Received

I2

Sciences, University of Milan, :Milan, Italy

June 1991.accepted 9 July 1991

We investigated the changes in P-adrenoceptor responses induced in guinea-pig tracheal and cardiac tissues by the anaphylactic reaction. Antigen aerosol challenge in sensitized guinea-pigs resulted in a marked reduction in adrenaline relaxation in isolated trachea ex vivo. The isoprenaline effect was also slightly decreased by antigen exposure, suggesting a possible impairment of tracheal &adrenoceptor function. On the other hand, the chronotropic and inotropic activity of adrenaline in isolated atria was not modified by the anaphylactic shock. suggesting a specific involvement of lung /3-adrenoceptors in the allergic reaction. Adrenaline

responses; j3-Adrenoceptors;

1. Introduction The activation of lung P-adrenoceptors leads to a potent bronchodilating effect which provides an important inhibitory mechanism of airway smooth muscle tone (Goldie, 1990). Impaired /3-adrenoceptor function was originally proposed to be a fundamental cause in the genesis of asthma pathology (Szentivanyi, 1968). Indaed a reduced number of /3-adrenoceptors and an impaired responsiveness to P-agonists have been described in asthmatic patients (Goldie, 1990). Even if general opinion now indicates that any j?-adrenoceptor down-regulation in asthma is likely to be secondary to the asthmatic processes, P-adrenoceptor hypofunction may contribute to further deterioration of this disease (Lulich et al., 1988; Goldie, 19901. Changes in adrenoceptor number have already been demonstrated by Barnes et al. (1980) in a guinea-pig model of chronic asthma. Therefore we investigated from a functional point of view the possible changes in P-adrenoceptor responsiveness induced in actively sensitized guinea-pig exposed to aerosolized antigen. In particular, we studied the responses elicited by the

Anaphylaxis: Lung (guinea-pig)

physiological adrenergic mediator, adrenaline, monary and cardiac tissues in vitro.

in pul-

2. Materials and methods Male Dunkin-Hartley guinea-pigs (350-451, g; Rodentia, Italy) were actively sensitized to ovalbumin by injecting 100 mg kg-’ i.p. and S.C.of this protein. The animals were anesthetized with urethane (1.3 g kg-’ i.p.) 18-28 days after sensitization and were mechanically ventilated (Rodent ventilator, Basile. Italy; SO strokes min- ’ of 1 ml room air per 100 g body weight). Spontaneous breathing was suppressed with pancuronium bromide (2 mg kg-’ i.m.1. Pulmonary inflation pressure (PIP, mm Hg) was measured with a pressure transducer (Bentley Trantec 800) connected to the ventilator circuit and the signals were displayed on a Basile (705 1) pen recorder. After 10 min stabilization, the animals were challenged by direct inhalation either with ovalbumin aerosol (10 mg ml-’ for 5 s) or with carbachol aerosol (3 mg ml-’ for 5 s), both generated by a DeVilbiss Pulmosonic Ultrasonic nebulizer (Daffonchio et al.. 1988). Sensitized animals not challenged with ovalbumin were considered as the control group. All the animals were left on mechanical ventilation for an additional hour, then killed by exsanguination. The tracheae mounted

were dissected. cut into zig-zag strips and in 20-ml organ baths for the measurcmcnt of

changes

in their Icnglh. as already

reported

(Daf-

fonchio et al., 19901. Log-concentration response curves for adrenaline and isoprenaline were obtained with tracheas maximally contracted with pilocarpine (20 PM). Antigen exposure did not modify pilocarpine-induced contraction, which was 117.8 + 15.9 and 97.2 t16.4 mm in control and antigen challenged tissues. respectively. In other experiments, the heart was rapidly removed from control and ovalbumin-challenged guinea-pigs. The right and left atria were separated intact from the ventricles, were grossly cleaned of excess fat and vessels and transferred to the organ bath containing oxygenated Ringer solution (34 * CI. The tissues were connected to isometric transducers (Basile) at a resting tension of 3 g. Changes in the contractile force and spontaneous beating induced by adrenaline were recorded on a Basile (7070) pen recorder. The data are reported as means f S.E.M. of (n) replications. Statistical comparisons between the log concentration-response curves were performed by a computer-aided program based on the parallel line assay (Snedecor and Cochran, 1967), which allows calculation of the dose ratio (DRl and its 95% confidence limits tconf. lim.). A probability level of P < 0.05 was considered statistically significant.

3. Results Direct inhalation of the antigen (ovalbumin, 10 mg ml-’ for 5 s) in actively sensitized and anesthetized guinea-pigs induced a sustained bronchoconstriction (50.53 + 5.32 mm Hgl. The anaphylactic reaction was followed by a marked reduction in adrenaline relaxing capacity tested ex vivo in isolated guinea-pig trachea. As shown in fig. 1, log-con~entratio,l response curves for the adrenergic mediator obtained with tissues taken from antigen-challenged animals (n = 8) were significantly (P < 0.01) shifted to the right of curves obtained in control tracheas tn = 71, with a DR of 3.92 (95% conf. lim, 2.38-6.45). Impaired adrenaline-mediated relaxation was not a consequence of the bronchoconstriction per se, since no change in the relaxing capacity of adrenaline was observed in the tracheal tissues when compared to the controls (DR 1.18, 95% conf. lim. 0.82-1.70; n = 8; fig. 1) after carbachol administration (3 mg ml-’ for 5 sl and despite an increase in PIP similar to that obtained with the antigen (51.94 F 6.16 mm Hgl. Also, the relaxing capacity of isoprenaline was significantly (P < 0.011, if only slightly reduced after the anaphylactic reaction. Log concentration-response curves for isoprenaline in the tracheas taken from ovalbumin-challenged animals were shifted to the right when compared to controls with a DR of 1.39 (95% conf. lim. 1.17-1.65; n = 6 for each group). In spontaneously beating atria, the anaphylactic reacti w triggered in vivo slightly reduced the actual

6

7

adrenaline

5

6

-log

M

Fig. 1. Log concentration-response curves for the relaxing activity of adrenaline tested in isolated guinea-pig tracheas taken from control animals (closed circle) and animals exposed to antigen (ovalbumin aerosol, 10 mg ml-’ for 5 s; open circle) or carbachol (aerosol, 3 mg ml-’ for 5 s; open triangle). The data are expressed as Cr, of adrenaline-induced maximal relaxation and are shown as the means kS.E.M. from at least seven replications (S.E.M. bars are not shown when they are contained within the symbol). The anaphyIa~ie reaction significantly (P < 0.01) shifted to the right the adrenaline log concentration-response curve (DR = 3.92, 95% conf. lim. 2.38-6.45). The maximal relaxation induced by adrenaline was 102.3k 17.0, 91.1 zt5.8 and 91.1 k9.9 mm in tissues taken from control guinea-pigs, and guinea-pigs challenged with antigen or carbachol, respectively.

baseline frequency from 162.5 rt: 13.6 (control, n = 4) to 134.2 + 13.6 (antigen challenged, n = 6) beats/min. However, antigen exposure did not modify the chronotropic and the inotropic effects of adrenaline. Log concentration-response curves calculated for the % increase in frequency and contractiIe force induced by adrenaline over the basal values were similar in the preparation from control and ovalbumin-challenged animals. The DR values were 1.40 (95% conf. lim. 0.40-4.86) and 1.22 (95% conf. lim. 0.43-3.47) for the chronotropic and inotropic effects, respectively.

4. Discussion The autonomic adrenergic system, through the activation of lung /3-adrenoceptors, plays an important role in the regulation of airway calibre. Considerable evidence supports the presence of abnormalities of the sympathetic drive in asthma pathology, possible as a consequence of the disease itself (Lulich et al., 1988; Goldie, 1990). Our data confirm and extend these findings, showing an impairment of adrenaline-mediated relaxation induced by the anaphylactic bronchoconstriction. This phenomenon seems to be specifically induced by the release of the anaphylactic mediators, since a similar bronchoconstriction caused by carbachol exposure did not modify the relaxing capacity of adrenaline. Even if the isoprenaIine relaxing capacity was only slightly reduced following antigen exposure, an overall impairment of lung P-adrenoceptor function can be suggested. Indeed, P-adrenoceptor down-regulation (reduced /3-adrenoceptor number) has been

99

shown to occur in guinea-pig pulmonary tissues following both in vitro and in vivo antigen challenge (Daffonchio et al., 1990; Barnes et al., 1980). The discrepancy between functional effects of isoprenaline and adrenaline is difficult to explain. However, isoprenaline has been demonstrated to be less susceptible to the desensitization procedure performed in sensitized guinea-pig trachea (Daffonchio et al., 1989). Thus, the minor decrease in isoprenaline-mediated relaxation compared to that with adrenaline might depend on their different pharmacological profiles or on a greater reserve and pulmonary /?-adrenoceptors for isoprenaIine than for adrenaline. The hypersensitivity reaction is known to affect cardiac responses. However, these alterations do not seem to involve impairment of cardiac /3-adrenoceptors, since the % increases in frequency and contractile force induced by adrenaline in the isolated atria were not modified by ovalbumin challenge. It is tempting to speculate that lung ~-adrenoceptors are in some way more sensitive to the mediators released during the anaphylactic bronchoconstriction. The different receptor subtypes and their transductional mechanisms present in lung (mainly &I or cardiac (mainly p,) tissues might account for this discrepancy. Moreover, there might be a larger reserve of cardiac ~~-adrenoceptors than of lung &-adrenoceptors. On the other hand, the experimental procedure used might also explain our negative results on the atria, since the aerosol challenge has been shown to induce a more selective bronchial response with reduced systemic effect (Daffonchio et al., 1987). Therefore, the anaphylactic reaction triggered within the lung might affect the adrenaline response only at the P-adrenoceptors present locally. Nevertheless, whatever mechanism is involved, these data underline a selective interference of the anaphyIa~tic mediators with the adrenaline response within the airways, a finding which might be relevant to asthma, taking into account that antigen exposure in asthmatics occurs predominantly by the inhalation route. In conclusion, our data indicate clearly that antigen exposure can lead to a reduction in airway P-adrenoceptor function. The response to adrenaline is particularly affected, indicating a peculiar alteration in the

inhibitory control of airway smooth muscle provided by the physiological /3-adrenergic mediator. This phenomenon, even if it may not account for the genesis of asthma, might contribute to the deterioration of symptoms and particularly to the development of airway hyperreactivity. In this regard, the degree of bronchial responsiveness in asthmatic subjects correlates with changes in @-adrenoceptor number and P-adrenoceptar-mediated responses ~Motojima et al., 1983). Moreover, airway hyperreactivity has been demonstrated to occur at the same time after the anaphylactic reaction triggered by antigen aerosol challenge in this experimental model (Daffonchio et al., 1988).

References Barnes, P.J.,

CT. Dollery and J. MacDermont, 1980, Increased pulmona~ a-adrenergic and reduced ~-adrenergic receptors in experimental asthma, Nature ,285, 569. Daffonchio, L., 1.W. Lees, A.N. Payne and B.J.R. Whittle, 1987. Pharmacological modulation of bronchial anaphylaxis induced by aerosol antigen challenge in anesthetized guinea-pigs, Br. J. Pharmacol. 91,701. Daffonchio. L., A.N. Payne, I.W. Lees and B.J.R. Whittle, 1988. Immediate anaphylactic bronch~nstriction induced airway hy~rreactivity in anaesthetized guinea-pigs. Br. J. Pharmacol. 94, 663. Daffonchio, L., A. Hernandez. G. Brunelli and C. Omini, 1989, Active sensitization modifies /%adrenoceptor reactivity in guineapig trachea, Pulmonary Pharmacol. 1. 161. Daffonchio, L., M.P. Abbracchio, M. Di Luca, A. Hernandez. L. Amadeo, F. Cattabeni and C. Omini. 1990, ~-Adren~eptor desensitization induced by antigen challenge in guinea-pig trachea, Eur. J. Pharmacol. 178, 21 Goldie, R.G., IYYO, Receptors in asthmatic airways, Am. Rev. Respir. Dis. 141, 151. Lulich, K.M., R.G. Goldie and J.W. Paterson, 1988, p-Adrenoceptor function in asthmatic bronchial smooth muscle, Gen. Pharmacol. 19, 307. Motojima, S., T. Fukuda and S. Makino, 1983. Measurement of p-adrenergic receptors on lymphocytes in normal subjects and asthmatics in relation to @-adrenergic hyperglycaemic response and bronchial responsiveness,Allergy 38. 331. Snedecor, G.V. and W.G. Cochran, 1967, Comparison of regression lines, in: Statistical Methods (The Iowa State University Press VI. Ames) p. 432. Szentivanyi, A., 1968, The P-adrenergic theory of the atopic abnormality in bronchial asthma, J. Allergy 42. 203.

Adrenaline-induced tracheal relaxation ex vivo is depressed following aerosol antigen challenge of sensitized guinea-pigs.

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