Introduction

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NKA and SP are tachykinin neuropeptides that have a number of potentially important effects on airway function including airway smooth muscle contraction, vasodilatation, airway microvascular leakage, mucus hypersecretion and potentiation of cholinergic neurotransmission (Maggi et al., 1995). Additionally, tachykinins have a number of proinflammatory effects and cause degranulation of mast cells and recruitment and activation of polymorphonuclear leukocytes and lymphocytes (Calvo et al., 1992; Bost and Pascual, 1992; Kähler et al., 1993; DeRose et al., 1994; Joos et al., 1994). These findings indicate that tachykinins may be involved in the pathogenesis of asthma.

Three tachykinin receptors have been pharmacologically identified (NK₁, NK₂, and NK₃ receptors) (Maggi et al., 1995). Activation of both NK₁ and NK₂ receptors produces bronchoconstriction in guinea pigs (Regoli et al., 1988; Ireland et al., 1991; Maggi et al., 1991; Ellis et al., 1993) although in other species such as the hamster (Maggi et al., 1989; Ellis et al., 1993), rabbit (Sheldrick et al., 1990) and humans (Ellis et al., 1993; Sheldrick et al., 1995) the contractile response to tachykinins is mediated predominantly by NK₂-receptor stimulation. NK₃ receptor activation increases excitability of the parasympathetic nervous system in guinea pigs (Myers and Undem, 1993) and this may contribute to augmented cholinergic hyperresponsiveness to tachykinins in this species. Tachykinins also constrict airway smooth muscle in dogs. Shioya et al. (1995) found that the dual NK₁/NK₂NK₂-receptor antagonist, FK 224, inhibited the contractile response of canine airway smooth muscle to SP and NKA. However, the functional role of NK₁ and NK₂ receptors on airway smooth muscle contractility cannot be ascertained from this study because selective NK₁ and NK₂ antagonists were not used. Tachykinins have a variety of effects on airway function in dogs and cause mucus gland hypersecretion (Coles et al., 1984; Haxhiu et al., 1991), stimulate tracheal ciliary beat frequency (Wong et al., 1990, 1991), promote chloride flux and modulate transmucosal potential difference across tracheal epithelium (Al-Bazzaz et al., 1985; Rangachari et al., 1987) and cause vasodilation of bronchial and pulmonary arteries (McCormack et al., 1989). Surprisingly, the in vivo effect of tachykinins on bronchomotor tone in dogs has not been previously studied.

In our study, we investigated the effect of NKA on airway mechanics and respiration in spontaneously breathing, anesthetized male beagle dogs. We also performed studies with the NK₁-receptor antagonist, CP 99994 (McLean et al., 1993) and the NK₂-receptor antagonist, SR 48968 (Emonds-Alt et al., 1992; Advenier et al., 1992) to determine the role of these tachykinin receptors on responses to NKA. Furthermore, we measured responses to NKA in dogs that were treated with the anticholinergic drug, ipratropium bromide (Pakes et al., 1980), to evaluate the role of cholinergic reflexes.

	Materials and Methods

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Animal preparation. Studies were performed on spontaneously breathing, male beagle dogs ranging in weight from 10 to 15 kg. The dogs were fasted overnight but given water ad libitum. The front paw was shaved and a 22 gauge Surflo catheter (Terumo Medical Corp., Elkton, MD) was inserted into the cephalic vein and secured in place with adhesive tape. A luer-lock Surflo injection plug (Terumo Medical Corp., Elkton, MD) was connected to the i.v. catheter to facilitate the administration of drugs. An i.v. drip of isotonic saline (0.9%, pH 5.6) was maintained throughout the experiments. Anesthesia was induced by the i.v. injection of sodium thiopental (25 mg/kg). Occasionally, a supplemental bolus of sodium thiopental (5 mg/kg, i.v.) was given just before the start of the experiment.

Pulmonary measurements. A cuffed endotracheal tube (Rüsch AG, Waiblingen, Germany; size 7.0 mm) was inserted into the trachea with the aid of a laryngoscope. The endotracheal tube was connected to a heated pneumotachograph (Hans Rudolph Inc., Kansas City, MO; model 3719, Flow 0-100 liter/min) and the pressure drop across the pneumotachograph was measured with a differential pressure transducer (Validyne, Northridge, CA; model MP 45-14-871, range ± 2 cm H₂O) and used to derive the measurement of . The airflow signal was converted to an electrical signal proportional to the V_T with an integrator circuit (Buxco Electronics Inc., Sharon, CT, model 6). A balloon-tipped esophageal catheter was placed into the esophagus and positioned at the point where recorded inspiratory pressure was greatest. Ptp was measured with a differential pressure transducer (Validyne, Northridge, CA; model MP 45-24-87, range ± 20 cm H₂O) connected to the esophageal balloon and to an air port in front of the endotracheal tube.

Aerosol challenge. A three-way breathing valve was interposed between the pneumotachograph and the endotracheal tube to facilitate the pulmonary delivery of aerosols. A Raindrop jet nebulizer (Puritan Bennett, Lenexa, KS) was used to generate aerosols that were delivered with 40 psi of compressed air at a flow of 150 ml/sec. Each challenge with the aerosolized drug consisted of five separate forced inflations of 4-sec duration per inflation (600 ml of air/inflation) that was given over a 1-min period. During this period a one-way breathing valve (Hans Rudolph Inc., model 140) was connected to the end of the pneumotachograph and the exhaled gas was collected in a Douglas bag for disposal. Doses were altered by varying the concentrations of the solution in the nebulizer.

Experimental studies. Initially, to determine the dose-response and temporal effects of NKA challenge on lung mechanics, RL and C_Dyn were measured immediately before and 0.5, 1, 3, 5 and 10 min after challenge with NKA (0.1 and 1%). Comparisons were made in the same dogs after challenge with aerosolized saline. In all subsequent studies we used a 1% solution of NKA for the challenge. In one such study, lung mechanics (RL and C_Dyn) and ventilatory parameters (V_T and f) were measured immediately before and 0.5 min after challenge with NKA. This time was selected in this, and in subsequent experiments, to measure the peak bronchoconstrictor and ventilatory response to the challenge (see "Results").

Statistics. Statistical significance of treatment effects was assessed by repeated measures analysis of variance on log-transformed data. Pair-wise comparisons between treated and control groups were performed using t tests based on model-estimated S.E. Comparisons with P < .05 were considered to be evidence of significant treatment effects.

Drugs. Sodium thiopental was purchased from Abbott Labs. (Chicago, IL), NKA from Peninsula Labs. (Belmont, CA), ipratropium bromide from Sigma Chemical Co. (St. Louis, MO) and methacholine chloride from Aldrich Chemical Co. (Milwaukee, WI). SR 48968 and CP 99994 were synthesized at Schering-Plough Research Institute (Kenilworth, NJ).

Animal care and use. These experiments were performed with prior approval of the Animal Care and Use Committee of Schering-Plough Research Institute which is a facility accredited by the American Association for the Accreditation of Laboratory Animal Care.

	Results

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Response to NKA. The results of figure 1 illustrate the dose-response and temporal effects of NKA challenge on RL and C_Dyn. After challenge with NKA (0.1 and 1%) there was a dose-dependent increase in RL and decrease in C_Dyn that peaked at 0.5 min after the challenge. This effect lasted approximately 3 to 5 min after challenge with 1% NKA. By 10 min after the NKA challenge, the RL and C_Dyn had returned to baseline values. Upon challenge with aerosolized saline there was a transient (0.5-1 min duration) increase in C_Dyn with no change in RL over the 10-min period (fig. 1). Similar effects on C_Dyn were also seen after challenge with compressed air indicating that this response is a function of the lung hyperinflation (600 ml of air/inflation) induced by the challenge procedure.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Dose-response and temporal effects of NKA challenge on pulmonary mechanics. The percentage change in RL and CDyn were measured for 10 min after challenge with aerosolized NKA (0.1%) , (1%) 0, or saline, . Values represent mean ± S.E.M. (n = 6 for 1% NKA and saline, n = 3 for 0.1% NKA). * P < .05 compared to aerosol saline.

Effect of tachykinin antagonists. Pretreatment of dogs with oral SR 48968 (1-10 mg/kg, p.o.) dose-dependently inhibited the increase in RL and decrease in C_Dyn due to challenge with NKA (fig. 2). There was also a dose-dependent inhibition by oral SR 48968 of the decrease in V_T and increase in f due to NKA challenge (table 2). At doses of 3 and 10 mg/kg, V_T increased after the NKA challenge (table 2). This response is a function of the lung hyperinflation induced by the challenge procedure because an increase in V_T is also seen after challenge with compressed air. There was no change in baseline RL, C_Dyn, V_T or f after treatment with SR 48968. 

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of oral SR 48968 on the bronchoconstrictor response to challenge with NKA. Dogs were treated with SR 48968 2 hr before challenge with NKA (1%). Values represent the mean ± S.E.M. (n = 11 per dose). * P < .05 compared to vehicle control.

Effect of ipratropium bromide. When dogs were treated with aerosolized ipratropium bromide (0.01%) and challenged with NKA (1%), there was a partial reduction of the increase in RL after the NKA challenge (fig. 3). Statistically significant effects with ipratropium bromide were observed at 1 and 3 min after the NKA challenge. However, the reduction of C_Dyn due to NKA was not significantly changed by treatment with ipratropium bromide (fig. 3). Ipratropium bromide had no effect on the reduction of V_T and increase in f after NKA challenge (data not shown). Furthermore, ipratropium bromide alone had no effect on baseline RL, C_Dyn, V_T and f when assessed before the NKA challenge.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of ipratropium bromide on the bronchoconstrictor response to challenge with NKA. Dogs were treated with aerosolized ipratropium bromide (0.01%),  or aerosolized saline,  10 min before challenge with NKA (1%). Values represent the mean ± S.E.M. (n = 8 per dose). * P < .05 compared to aerosolized saline.

	Discussion

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NKA is a potent constrictor of airway smooth muscle and causes bronchospasm in rats (Joos et al., 1988; Joos and Pauwels, 1990), guinea pigs (Hua et al., 1984), monkeys (Mauser et al., 1997) and human asthmatics (Evans et al., 1988; Crimi et al., 1992; Joos et al., 1996). NKA is the preferred ligand for NK₂-receptors which is the predominant receptor subtype producing bronchoconstriction in most species, including humans (Maggi et al., 1995). In our study in dogs, we demonstrated that inhaled NKA produced bronchoconstriction that was blocked by NK₂-receptor antagonist SR 48968, but not by the NK₁-receptor antagonist CP 99994. These results identify the NK₂-receptor as the functionally relevant receptor subtype producing bronchospasm to NKA in dogs. We also performed a few experiments with aerosolized SP but found no bronchoconstrictor response after challenge. Only coughing was observed in some of the dogs. These findings suggest that the NK₁-receptor is not functionally important for producing bronchoconstriction in dogs.

Challenge with NKA produced an increase in lung resistance and a decrease in dynamic lung compliance. This change in pulmonary mechanics is typically seen with other bronchoconstrictor agents, such as methacholine chloride. The bronchoconstrictor response to NKA peaked at 0.5 min after challenge and had a duration of only 3 to 5 min. Tachykinins are rapidly metabolized by a variety of endopeptidases present in the lungs (Lilly et al., 1993) which would explain the relatively transient nature of the bronchoconstrictor response. Most of the dogs studied responded with a bronchospasm but there was a wide range in bronchial reactivity to this spasmogen. In some of the more reactive dogs, coughing was occasionally seen immediately after the challenge. It is important to note that our studies were performed in normal, healthy dogs that had no evidence of pulmonary inflammation or pulmonary dysfunction. In humans, bronchoconstrictor responses to NKA are greater in asthmatics compared to normals (Joos et al., 1987), suggesting that bronchoconstrictor responses to this spasmogen in dogs would be augmented in the presence of pulmonary inflammation.

Both NK₁ and NK₂ receptors are found in the lungs and in some species, like the guinea pig, both NK₁ and NK₂ receptor stimulation produce bronchoconstriction (Regoli et al., 1988; Ireland et al., 1991; Maggi et al., 1991; Ellis et al., 1993). In other species such as hamster (Maggi et al., 1989; Ellis et al., 1993), rabbit (Sheldrick et al., 1990) and humans (Ellis et al., 1993; Sheldrick et al., 1995), only NK₂-receptor stimulation mediates airway smooth muscle contraction. Tachykinins constrict airway smooth muscle in dogs and Shioya et al. (1995) found that the dual NK₁/NK₂-receptor antagonist, FK 224, inhibited the contractile response of canine airway smooth muscle to SP and NKA. The results from our study identify the NK₂-receptor as the functionally important receptor subtype. We found that pretreatment with SR 48968, a selective NK₂-receptor antagonist (Emonds-Alt et al., 1992; Advenier et al., 1992) blocked the increase in RL and decrease in C_Dyn in response to NKA challenge whereas CP 99994, a selective NK₁-receptor antagonist (McLean et al., 1993) had no effect. We used a dose of CP 99994 that completely blocked the hypotensive response to i.v. SP. This physiological response is an established pharmacological procedure for evaluating NK₁-receptor antagonists in dogs (McLean et al., 1996).

Tachykinin receptors and tachykinin-containing immunoreactive nerve fibers are widely distributed in the pulmonary system of dogs (Hisa et al., 1985; Rangachari et al., 1987; McCormack et al., 1989; Nohr and Weihe, 1991). In several species, such as the guinea pig (Watson et al., 1993; Hey et al., 1996), rabbit (Tanaka and Grunstein, 1984, 1986) and sheep (Corcoran and Haigh, 1992), tachykinin receptors are located on airway parasympathetic nerves and augment the release of acetylcholine from postganglionic nerve terminals causing exaggerated cholinergic bronchoconstrictor response. In our study, we found that the bronchoconstrictor response to NKA was partially blocked by ipratropium bromide. This result identifies a cholinergic component to the bronchoconstrictor response to NKA in dogs. It is interesting to note that the predominant effect of ipratropium bromide was on the increase in RL. These results imply that the cholinergic component of the NKA-induced bronchospasm in dogs involves effects on airway caliber or possibly on the tissue viscance of the lungs because both these elements contribute to the derivation of pulmonary resistance in dogs (Ludwig et al., 1989). In this regard, the parasympathetic innervation of the pulmonary system in dogs is predominantly in the central conducting airways of the trachea, bronchi and bronchioles (Richardson, 1979) which makes it likely that the cholinergic component of the NKA-induced bronchospasm was at this location of the tracheobronchial tree.

In addition to their effects on airway smooth muscle contractility, tachykinins also stimulate a variety of airway sensory nerves such as lung irritant receptors (Prabhakar et al., 1987), pulmonary "C" fibers (Prabhakar et al., 1987; Widdicombe 1995) and carotid bodies (Prabhakar et al., 1989; Cragg et al., 1994). Therefore, the ventilatory response to NKA seen in dogs likely involves a complex interplay between the direct effect of NKA on airway caliber-producing airflow obstruction and an indirect, reflex effect from airway sensory nerve stimulation. Our results suggest that both NK₁ and NK₂ receptors are involved in this response because the respiratory response to NKA challenge was inhibited by SR 48968 and CP 99994. It is likely that the NK₂-receptor component involves effects on airway caliber-producing airflow obstruction that in turn would activate the lung irritant receptors producing rapid, shallow breathing (Widdicombe, 1995). The NK₁-receptor component does not involve airway smooth muscle contraction and may stimulate pulmonary reflexes directly. Indeed, from our studies in dogs (unpublished observations J. E. Sherwood and R. W. Chapman), we have found that intravenous SP (100 ng/kg), has a profound effect on respiration, i.e., produced an increase in respiratory rate, a reduction in V_T and an increase in minute volume with no concomitant change in lung mechanics. In this study we also found that the respiratory response to SP was completely blocked by CP 99994 indicating that it is produced by activation of the NK₁-receptor. Although SR 48968 and CP 99994 are capable of inhibiting pulmonary reflexes by acting at the level of the central nervous system (Bolser et al., 1997), we consider this to be an unlikely scenario in our study because neither SR 48968 nor CP 99994 had an effect on baseline ventilation and neither drug affected the respiratory response to inhaled methacholine challenge in our dogs (J. E. Sherwood and R. W. Chapman, unpublished observations).

In conclusion, we have identified an NK₂-receptor-mediated bronchoconstrictor effect of NKA in dogs. Cholinergic reflexes play a small, but significant role in this response. Furthermore, both NK₁- and NK₂-receptors appear to be involved in the respiratory response to NKA challenge. These results demonstrate an effect of tachykinins on airway mechanics and ventilatory reflexes in dogs.