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Br. J. Pharmacol. (1992), 107, 481-487

Macmillan Press

Ltd, 1992

Pharmacological modulation of inhaled sodium metabisulphite-induced airway microvascular leakage and bronchoconstriction in the guinea-pig Tatsuo Sakamoto, Wayne Elwood, Peter J. Barnes & 'K. Fan Chung Department of Thoracic Medicine, National Heart and Lung Institute, Royal Brompton Hospital, London SW3 6LY 1 We have investigated the effects of chlorpheniramine, atropine and capsaicin pretreatment on inhaled sodium metabisulphite (MBS)-induced airway microvascular leakage and bronchoconstriction in anaesthetized guinea-pigs in order to clarify the mechanisms involved in these responses. The effects of frusemide and nedocromil sodium were also examined. 2 Lung resistance (RL) was measured for 6 min after inhalation of MBS (20, 40, 80 and 200 mM; 30 breaths), followed by measurement of extravasation of Evans blue dye into airway tissues, used as an index of airway microvascular leakage. MBS caused an increase in RL and leakage of dye at all airway levels in a dose-dependent manner. 3 Chlorpheniramine (1O mg kg-', i.v.), atropine (1 mg kg-', i.v.), their combination or inhaled nedocromil sodium (1O mg ml-', 7 min) had no effect against the airway microvascular leakage induced by 80 mM MBS (30 breaths). Capsaicin pretreatment (50 mg kg-', s.c.) caused a significant decrease in the leakage of dye in the main bronchi and inhaled frusemide (10 mg ml-', 7 min) also in the main bronchi and proximal intrapulmonary airway. 4 Chlorpheniramine, atropine, their combination, capsaicin pretreatment and frusemide, but not nedocromil sodium, inhibited significantly the peak RL induced by 80 mM MBS (30 breaths) by approximately 50%. 5 We conclude that a cholinergic reflex and neuropeptides released from sensory nerve endings may participate in the mechanisms of MBS-induced airway responses. Frusemide but not nedocromil sodium may have an inhibitory effect on these neural mechanisms. The inhibitory effect of nedocromil sodium against lower doses of MBS is not excluded. Keywords: Bronchoconstriction; capsaicin; cholinergic reflex; frusemide; histamine; microvascular permeability; nedocromil sodium; sodium metabisulphite; tachykinin

Introduction Sodium metabisulphite (MBS) is used as a food preservative and an antioxidant, and can induce bronchoconstriction when inhaled by patients with asthma and by atopic nonasthmatic subjects (Seale et al., 1988; Nichol et al., 1989; Dixon & Ind, 1990). The mechanisms by which MBS induces bronchoconstriction are not clear. There is circumstantial evidence that sulphur dioxide gas released from MBS solution may be the active ingredient causing bronchoconstriction (Fine et al., 1987; Hein et al., 1991). Both sulphur dioxideand MBS-induced bronchoconstriction are inhibited by nedocromil sodium (Dixon et al., 1987; Dixon & Ind, 1990) and sodium cromoglycate (Snashall & Baldwin, 1982; Tan et al., 1982; Myers et al., 1986; Dixon & Ind, 1990), although these observations do not indicate the mechanisms involved. MBSinduced bronchoconstriction in patients with asthma is inhibited to a lesser extent by muscarinic receptor antagonists (Dixon & Ind, 1988; Seale et al., 1988; Nichol et al., 1989) and is not inhibited by a histamine Hi-receptor antagonist (Dixon & Ind, 1988). These studies suggest that MBSinduced airway responses may involve mechanisms other than a cholinergic reflex or histamine release. In order to investigate further the potential mechanisms of MBS-induced bronchoconstriction, we have studied the effect of MBS aerosol in anaesthetized guinea-pigs. We have determined whether MBS can, in addition to causing bronchoconstriction (Lotvall et al., 1990), induce plasma exudation in

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the airway. We have examined the contribution of a cholinergic reflex and of histamine release. In addition, we have tested the hypothesis that part of the effect of MBS in the airway of the guinea-pig is mediated by non-cholinergic bronchoconstrictor pathways through the release of endogenous tachykinins (Lundberg & Saria, 1987; Djokic et al., 1989; Lotvall et al., 1991) by studying the effect of tachykinin depletion with capsaicin pretreatment (Lundberg et al., 1983; Buck & Burks, 1986). Nedocromil sodium, a drug used for the prophylaxis of asthma, and frusemide, a loop diuretic, have been shown to inhibit airway smooth muscle contraction induced by stimulation of non-cholinergic nerves in the guinea-pig in vitro (Elwood et al., 1991; Verleden et al., 1991). We studied the effect of these agents on MBS-induced airway effects in the guinea-pig in vivo.

Methods Animal preparation Female Dunkin-Hartley guinea-pigs were anaesthetized with an initial dose of urethane (1.5 g kg-') injected intraperitoneally. Additional urethane (1.0-1.5 g kg-') was given 30 min later to achieve an appropriate level of anaesthesia as evidenced by a disappearance of corneal reflex and withdrawal response to paw pinching. This level of anaesthesia was maintained throughout the experiment until the time when suxamethonium was administered. Body temperature (rectal) was maintained at about 36°C by placing the animals

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under a lamp. A tracheal cannula (8 mm length and 2.7 mm internal diameter) was inserted into the lumen of the cervical trachea through a tracheostomy, and secured with a suture. A polyethylene catheter was inserted into the left carotid artery to monitor systemic mean blood pressure (BP) and heart rate with a pressure transducer (PDCR 75 S/N 1560, Druck Ltd., U.K.). The right external jugular vein was cannulated for the administration of intravenous drugs or solutions.

Measurements of airway function Animals were placed in a supine position with an intratracheal cannula connected to a constant volume mechanical ventilator (Model 50-1718, Harvard Apparatus Ltd., Edenbridge, U.K.), and then given a single injection of suxamethonium (1-1.5 mg kg', i.v.) to prevent interference by spontaneous respiration. A tidal volume of 10 ml kg-' and a frequency of 60 strokes min-' were used. Lung resistance (RL) and transpulmonary pressure were measured as an index of airway function and monitored throughout the experiments. Transpulmonary pressure was measured with a pressure transducer (Model FCO 40; ± 1000 mmH2O; Furness Controls Ltd., Bexhill, U.K.) with one side attached to a catheter inserted into the right pleural cavity and the other side attached to a catheter connected to a side port of the intratracheal cannula. The ventilatory circuit had a volume of 20 ml. Airflow was measured with a pneumotachograph (Model F1L; Mercury Electronics Ltd., Glasgow, U.K.) connected to a transducer (Model FCO 40; ± 20 mmH2O; Furness Controls Ltd.). The signals from the transducers were digitized with a 12-bit analog-digital board (NB-MIO-016; National Instruments, Austin, TX, U.S.A.) connected to a Macintosh II computer (Apple Computer Inc., Cupertino, CA, U.S.A.) and analyzed with software (LabView; National Instruments), which has programmed to calculate instantaneously RL by the method of von Neergaard & Wirz (1927).

Measurement of microvascular leakage Vascular permeability was quantified by the extravasation of Evans blue dye, which has been shown to correlate well with the extravasation of radiolabelled albumin in guinea-pig airways (Rogers et al., 1989). Six min after inhalation of MBS or its vehicle, the thoracic cavity was opened, and a cannula was inserted into the aorta through a ventriculotomy. Perfusion was performed with 100-150 ml 0.9% w/v sodium chloride in water (0.9% saline) at a pressure of 100-120 mmHg in order to remove intravascular dye from the systemic circulation. Blood and perfused liquid were expelled through incisions in the right and left atria. Subsequently, the right ventricle was opened, and the pulmonary circulation was perfused with 30 ml of 0.9% saline. The lungs were then removed, and the connective tissues, vasculature and parenchyma were gently scraped off with a blunt scalpel until

bronchial tissue was left. The tracheal portion from 6 to 13 mm distal to a tip of the tracheal cannula (TR) was collected and the main bronchi (MB) was sectioned off at a point 3 mm distal to the carina. The rest of the bronchial tract was divided into two components, arbitrarily termed 'proximal intrapulmonary airway (PIPA, the proximal 5-7 mm portion)' and 'distal intrapulmonary airway (DIPA, the remaining distal portion)'. The tissues were blotted dry, and then weighed. Evans blue dye was extracted in 2 ml of formamide at 40°C for 24 h, and measured in a spectrophotometer (Model 8480, Philips, Cambridge, U.K.) at 620 nm. The tissue content of Evans blue dye was calculated by interpolation on a standard curve of dye concentrations in the range of 0.5-10 jg mlh' and expressed as ng of dye mg1' of wet tissue.

Protocol Sodium metabisulphite challenge MBS dissolved in 0.9% saline was stored at - 20°C at a concentration of 1 M, and MBS solution (diluted in 0.9% saline) was prepared 24 h before experimentation. Three milliliters of the solution was then kept in a closed, air-tight container with a total capacity of 8 ml at room temperature. The container was shaken 5 times before the MBS solution was placed in an ultrasonic nebulizer (PulmoSonic Model 2511: Devilbiss Co., Somerset, PA, U.S.A.), from which MBS aerosol was generated and was administered to the airways for 30 breaths through a separate ventilatory system that bypassed the pneumotachograph. An additional five breaths was given through the nebulizer circuit to clear the system of aerosol. The volume of the circuit distal to the nebulizer was 20 ml. The output from the nebulizer at the port of the tracheal cannula, measured with airflow of 0.3 1 min-' (3 ml of 0.9% saline in the nebulizer), was 70 ± 5 fLl min' . The mean particle size was 3.8 jLm, with a geometric standard deviation of 1.3, measured with a laser droplet and particle analyzer (Model 2600C; Malvern Instruments, Malvern, U.K.).

Effect of different doses of sodium metabisulphite Animals weighing 440-500 g were divided into five groups (n = 5) in order to study the effects of the different concentrations of MBS (20, 40, 80 and 200 mM, 30 breaths) on RL and dye leakage in the airway, and were compared to those of the diluent for MBS. Ten min after connection to the ventilator, Evans blue dye (20 mg kg-', i.v.) was given for a period of 1 min. One min later, each concentration of MBS or 0.9% saline was inhaled for 30 breaths. RL was recorded every 30s. At 5 min after the administration of MBS or 0.9% saline, the animals were hyperinflated with twice the tidal volume by manually blocking the outflow of the ventilator. One min later, recovery RL was measured, and then the animals were perfused. Baseline RL was determined just before challenge. In addition, the airway effects induced by 80 mM MBS and 0.9% saline were compared to those induced by 80 mM MBS prepared just before use (n = 5), which caused a significant increase in RL with maximum of 2.04 ± 0.56 cmH2O mlh' s' and dye leakage only in MB (44.9 ± 4.1 ng mg-' of tissue). Both these responses were significantly smaller than those by the MBS prepared 24 h before use. The pH of 80 mM MBS prepared just before use was 2.93 ± 0.006, and the other was 2.71 ± 0.007 (P

Pharmacological modulation of inhaled sodium metabisulphite-induced airway microvascular leakage and bronchoconstriction in the guinea-pig.

1. We have investigated the effects of chlorpheniramine, atropine and capsaicin pretreatment on inhaled sodium metabisulphite (MBS)-induced airway mic...
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