INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Acute inhalation of cigarette smoke (CS) induces a transient bronchoconstrictive effect in humans, both smokers and nonsmokers alike. Recent studies carried out in our laboratory have shown that acute bronchoconstrictive effect of inhaled CS is mediated primarily through the activation of bronchopulmonary C-fiber afferent endings by nicotine (1). Stimulation of these afferent endings is known to elicit reflex bronchoconstriction via the cholinergic pathway (5) and also to trigger the release from these endings of tachykinins, which can induce potent bronchoconstrictive effects (6).

Airway hyperresponsiveness, characterized by exaggerated bronchomotor responses to various nonallergic and nonspecific stimuli, is a prominent feature of bronchial asthma (9, 10). However, whether the acute bronchoconstrictive effect of inhaled CS is augmented in patients with preexisting bronchial hyperresponsiveness is not known. Guinea pigs actively sensitized with ovalbumin (Ova) provide a well-established animal model that has been used extensively for studying the pathogenic mechanisms and therapeutic treatments of bronchial hyperreactivity, because many of their pathophysiological features closely resemble those seen in asthmatic patients (11- 13). Recently, it has been reported that Ova sensitization increases the excitability of C neurons in the guinea pig nodose ganglia (14). Furthermore, the involvement of tachykinins in the airway hyperresponsiveness of Ova-sensitized animals has also been reported; for example, the augmented bronchoconstrictive effect caused by allergen challenge can be attenuated by capsaicin treatment, which depletes tachykinins from these nerve endings (15, 16), and, conversely, can be potentiated by pretreatment with phosphoramidon, which prevents the degradation of the endogenous tachykinins by inhibiting the enzyme activity of neutral endopeptidase (17, 18). However, findings reported by other investigators do not always support a significant role of endogenous tachykinins in the airway hyperresponsiveness induced by Ova sensitization (19, 20). In addition, more direct evidence of the involvement of tachykinins has not previously been established. The recent development of selective antagonists to neurokinin-1 (NK₁) and NK₂ receptors has made it feasible to address this question. The present study was designed to determine (1) if the bronchoconstrictive effect of inhaled CS is enhanced when airway hyperresponsiveness is induced by Ova sensitization, and (2) if so, whether an increase in endogenously released tachykinins is involved.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The procedures described subsequently were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (21), published by the National Institutes of Health, and were also approved by the University of Kentucky Institutional Animal Care and Use Committee.

Ovalbumin Sensitization

Young, male, pathogen-free Hartley guinea pigs (initial weight approximately 350 g) were sensitized to Ova by daily exposure to aerosolized Ova for 5 min, 5 d a week, for 2 wk. During the exposure, guinea pigs were placed in a Plexiglas chamber (36 × 24 × 20 cm), which was connected to an ultrasonic nebulizer (Model 100; Devilbiss, Somerset, PA) and placed under a negative-pressure exhaust hood. Ova/saline solution (wt/vol concentration: 1%) was nebulized with an output rate of 0.2 ml/min and a droplet size ranging from 0.5 to 30 µm. During the second week, diphenhydramine (8 mg) was injected intraperitoneally 1 h before each exposure to Ova to alleviate the bronchospasm caused by release of histamine during the exposure. A matching group of guinea pigs serving as the control group was exposed to saline aerosol in an identical manner.

Measurements of Lung Mechanics

One day after the last exposure to Ova aerosol, guinea pigs were anesthetized with chloralose (100 mg/kg, intraperitoneally) and urethane (500 mg/kg, intraperitoneally), and supplemental doses of the same anesthetics were administered whenever necessary to maintain abolition of the corneal and withdrawal reflexes. The trachea was cannulated just below the larynx via a tracheotomy. The animals were placed in a supine position, and were ventilated with a respirator (Harvard model 683; South Natick, MA) at a constant rate of 44 breaths/min and a tidal volume (VT) of approximately 8 ml/kg; the latter was adjusted in each animal to maintain the end-tidal CO₂ concentration (Novametrix model 1260; Wallingford, CT) at approximately 5%. The right jugular vein and the right carotid artery were cannulated for intravenous injections and for arterial blood pressure (ABP) measurement. A catheter for measuring intrapleural pressure (Ppl) was inserted into the intrapleural cavity via a surgical incision between the fifth and sixth ribs; this incision was subsequently sutured and further sealed air-tight with silicone jelly. The pneumothorax was then corrected by briefly opening the intrapleural catheter to ambient air during a held hyperinflation (3 × VT). During the experiment, the animals were paralyzed with pancuronium bromide (30 µg/kg, intravenously). A heating pad was placed under the animal to maintain body temperature at approximately 36° C.

Transpulmonary pressure was measured as the difference between the tracheal pressure (Pt) and Ppl with a differential pressure transducer (Validyne MP 45-28; Northridge, CA). Respiratory flow was measured with a heated pneumotachograph and a differential pressure transducer (Validyne MP 45-14). All signals were recorded on a chart recorder (Grass model 7; Quincy, MA); total pulmonary resistance (RL) and dynamic lung compliance (Cdyn) were analyzed continuously by an on line computer on a breath-by-breath basis (TS-100 series; Biocybernetics; Taipei, Taiwan).

Acetylcholine (ACh) Dose-response Curve

To verify airway hyperresponsiveness, dose-response curves of RL and Cdyn to bolus injections (0.2 ml vol) of ACh (1.0 to 5.06 µg/kg) were determined in each animal by successively increasing the concentration of ACh solution by 50% at 5-min intervals. The lungs were hyperinflated (3 × VT) at 2 min before each ACh injection.

Cigarette Smoke Inhalation Challenge

Smoke (7.5 ml) was generated from the midportion of a lighted cigarette by a smoke machine, mixed with an equal volume of room air, and delivered directly into the lung via the inspiratory line of the respirator over approximately four consecutive respirator cycles (3). The lungs were hyperinflated (3 × VT) periodically and also at 2 min before each CS challenge to maintain a constant volume history. The cigarettes used in this study were the University of Kentucky Research Series 2R1, containing 2.45 mg of nicotine and 35.3 mg of tar per cigarette. At least 30 min elapsed between two CS challenges to avoid tachyphylaxis.

Allergic Skin Reaction (ASR) Test

To test whether treated animals were sensitized to Ova, the back of each guinea pig was shaved for ASR test. Through a 27-gauge skin-test needle, 0.03 ml of each of the following solutions was injected into the upper dermis intradermally: isotonic saline, histamine (30 µg), and Ova (0.5, 1.0, and 5 µg). To quantitatively evaluate the response, two perpendicular diameters of the resulting wheal were measured 60 min after the injection, and the average was then calculated.

Inflammatory Cell Analysis in Bronchoalveolar Lavage Fluid (BALF)

BALF was obtained by injecting 10 ml of sterile saline (5 ml, twice) via the tracheal cannula. The collected BALF (approximately 7 ml) was centrifuged at 1,500 rpm for 10 min, and the pelleted cells were treated with TRIS-buffered ammonium chloride solution (pH 7.2) to lyse red blood cells. The remaining cells were washed once with phosphate-buffered saline supplemented with 1% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin (GIBCO, Grand Island, NY). Total cell counts were determined by using a hemocytometer. Differential leukocyte counts were then performed on cytospin slides. A minimum of 500 leukocytes were counted by using standard morphologic criteria.

Radioimmunoassay (RIA) of Neuropeptides

After supernatant fractions of the collected BALF had been desalted, purified with C₁₈ Sep-Pak columns, lyophilized, and reconstituted in RIA buffer, substance P-like immunoreactivity (SP-LI) and calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) were measured by RIA, as previously described (1).

Experimental Protocol

Three series of experiments were carried out. Study Series 1 was carried out to determine whether the bronchomotor response to inhaled CS was altered by Ova sensitization. The dose-response curve to ACh was obtained in each animal; the responses were later compared between control (n = 9) and Ova-sensitized (n = 9) animals for verifying whether airway hyperresponsiveness was developed in the Ova-sensitized animals. Subsequently, responses of RL and Cdyn were measured continuously in each animal for 1 min before and for 2.5 min after CS inhalation, and the challenge was repeated > 30 min later to test the reproducibility of the response. Study Series 2: To determine the possible involvement of endogenous tachykinins, the responses to CS inhalation challenges were determined in a separate group of Ova-sensitized guinea pigs before and after pretreatment with either CP-99994 (0.3 mg/kg, intravenously; n = 7), a selective antagonist of NK₁ receptors, or SR-48968 (0.3 mg/kg, intravenously; n = 6), a selective antagonist of NK₂ receptors, and again after the combination of both of these two compounds (n = 13); the doses of these antagonists were determined on the basis of previous findings (2). To determine a possible involvement of the cholinergic pathway, the responses to inhaled CS were determined in another group of Ova-sensitized guinea pigs before and after pretreatment with atropine (0.05 mg/kg, intravenously; n = 5). We also tested whether pretreatments with these two NK-receptor antagonists changed the bronchomotor response to ACh in six additional guinea pigs sensitized with Ova; the dose of ACh (5.06 µg/ kg) was chosen because it induced a similar degree of bronchoconstriction as the inhaled CS. Study Series 3: Airway inflammation was determined by differential leukocyte counts in the BALF; results were compared between control (n = 10) and Ova-sensitized animals (n = 10). Tachykinin and CGRP releases induced by CS inhalation challenge were also determined in two matching groups of animals (control, n = 10; Ova, n = 10), and each group was randomly divided into two subgroups (n = 5 each); BALF was obtained at the baseline (before smoke) in one subgroup and within 1 min after the CS inhalation challenge (15 ml; 100% concentration) in the other subgroup.

Statistical Analysis

In each experiment, we chose the six consecutive breaths immediately before the CS challenge as the baseline, the six consecutive breaths with peak increase in RL that occurred within 20 breaths after the CS challenge as the first-phase response, and breaths 75 to 80 as the second-phase response. A two-way analysis of variance (ANOVA) was used for the statistical analysis; one factor was the treatment effect of Ova or NK-receptor antagonists, and the other factor was the effect of inhaled CS. When the two-way ANOVA showed a significant interaction, pairwise comparisons were made with a post hoc analysis (Fisher's least significant difference). Data are reported as means ± SEM. A p value < 0.05 was considered significant.

Materials

Pancuronium bromide (Genesia Pharm., Inc., Irvine, CA) acetylcholine chloride (Sigma, St. Louis, MO), atropine sulfate (Sigma), diphenhydramine (Sigma), and CP-99994 (Pfizer Inc., Groton, CT) were diluted in isotonic saline. SR-48968 (Sanofi Recherche, Montpelliex Cedex, France) was first dissolved in polyethylene glycol (average molecular weight: 200; Sigma) and then diluted in saline at a 1:1 ratio to a final concentration of 0.67 mg/ml.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Series 1: Effect of Ova Sensitization on the Bronchomotor Response to Cigarette Smoke

After daily exposure to aerosolized Ova for 2 wk, the average weight of the animals was 498 ± 6 g (n = 9), which was not significantly different from that of the control animals exposed to saline aerosol (506 ± 10 g; n = 9). The results obtained from the skin test (Table 1) showed distinct differences between the Ova-sensitized and control groups in their reactions to all three doses of Ova injections, whereas no difference was found in their reactions to histamine injections. Neither group showed any positive skin reaction to saline injection.

There was no significant difference in the average baseline RL or Cdyn between control and Ova-sensitized animals, but the dose-response curve to ACh was significantly elevated in the latter (Figure 1), verifying the presence of nonspecific airway hyperresponsiveness in the Ova-sensitized group (22). The bronchomotor response to CS was also markedly augmented in Ova-sensitized animals (Figure 2). In control animals, inhalation of CS triggered only a very mild bronchoconstriction, and there was no clear initial response (Figure 3); the only significant change from the baseline values was a mild delayed reduction in Cdyn (Figure 4). By contrast, in sensitized animals the same CS inhalation challenge evoked a rapid and intense increase of the RL, which increased from a baseline of 0.16 ± 0.02 cm H₂O/ml/s to a peak of 0.84 ± 0.08 cm H₂O/ml/s in < 15 s, and then started to decline toward but remained slightly above the baseline (Figure 3). Cdyn also decreased rapidly from a baseline of 0.87 ± 0.09 ml/cm H₂O to 0.46 ± 0.10 ml/cm H₂O after the CS inhalation challenge, but remained at that low level even after RL had almost completely recovered (Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Dose responses of RL and Cdyn to intravenous injections of ACh in control and Ova-sensitized guinea pigs. Peak response to each dose of ACh in each animal was averaged over three consecutive breaths that occurred within 10 breaths after the injection. Open circles: responses in control animals; filled circles: Ova-sensitized animals. Data represent means ± SEM of nine guinea pigs in each group. *Significant difference comparing corresponding data between control and Ova-sensitized groups.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Experimental record illustrating the responses of transpulmonary pressure (Ptp), respiratory flow ( ; inspiratory flow: positive), and ABP to cigarette smoke in a control (weight: 530 g) and an Ova-sensitized guinea pig (weight: 510 g). Cigarette smoke (15 ml, 50%) was delivered into the lung by respirator in four consecutive breaths (between the two arrows).

View larger version (31K): [in this window] [in a new window]   Figure 3.   Effect of Ova sensitization on the bronchomotor responses to acute inhalation of cigarette smoke (15 ml, 50% concentration). Triangles: responses to smoke inhalation challenge in control animals; circles: responses in Ova-sensitized animals. Reproducibility of the responses was tested in two consecutive challenges in each animal. Open symbols: the first smoke inhalation challenge; closed symbols: the same challenge repeated 30 min later. Cigarette smoke was delivered into the lung by respirator between the two arrows. Data represent means of nine guinea pigs in each group; to avoid clutter, SEM is shown only for every fifth breath number. There is no statistical difference between the responses to the first and second cigarette smoke challenges in either group.

View larger version (20K): [in this window] [in a new window]   Figure 4.   Comparison of the bronchomotor responses to cigarette smoke between control and Ova-sensitized guinea pigs. Open bars: baselines averaged over six consecutive breaths immediately before the smoke inhalation; filled bars: first-phase responses averaged over six consecutive breaths with peak increase in RL that occurred within 20 breaths after the smoke inhalation; hatched bars: second-phase responses averaged over breaths 75 to 80 after the smoke inhalation. Data represent means ± SEM of nine guinea pigs in each group. *Significantly different from baseline; significant difference comparing corresponding data between control and Ova-sensitized guinea pigs.

Study Series 2: Role of Tachykinins

This series was carried out only in Ova-sensitized animals, subdivided into two groups. In the first group (n = 7), CS inhalation challenge evoked a bronchoconstrictive response similar to that shown in Study Series 1. Fifteen minutes after the administration of CP-99994 (0.3 mg/kg, intravenously) (45 min after the previous CS inhalation challenge), the CS challenge was repeated, but the response of RL to CS was not significantly altered (Figure 5). However, the addition of SR-48968 (0.3 mg/kg, intravenously) almost completely blocked the CS-induced bronchoconstriction in these animals (Figure 5). In the second group (n = 6), administration of SR-48968 alone eliminated more than 85% of the CS-induced bronchoconstriction, and the remaining response was almost completely abolished after addition of CP-99994 (Figure 5); the only response to CS that persisted after the administration of SR-48968 and CP-99994 in combination was a slight reduction of Cdyn during the second phase (Figure 6). In contrast, pretreatments with CP-99994 and SR-48968 did not significantly reduce the bronchomotor response to ACh (5.06 µg/kg, intravenously) in six other guinea pigs sensitized with Ova (Figure 7). In a separate group of Ova-sensitized guinea pigs (n = 5), pretreatment with atropine did not significantly alter the peak bronchomotor response to inhaled CS (before atropine, RL = 0.53 ± 0.07 cm H₂O/ml/s; after atropine: RL = 0.44 ± 0.08 cm H₂O/ml/s; p > 0.05).

View larger version (33K): [in this window] [in a new window]   Figure 5.   Effect of pretreatments with CP-99994 and SR-48968 on the bronchomotor responses to cigarette smoke in Ova-sensitized guinea pigs. (Upper panel [n = 6]) Open circles: responses to smoke during control; filled circles: responses at 15 min after pretreatment with CP-99994 (45 min after the previous smoke inhalation challenge); open triangles: responses at 15 min after pretreatments with CP-99994 and SR-48968. (Lower panel [n = 7]) Open circles: responses to smoke during control; filled circles: responses at 15 min after pretreatment with SR-48968; open triangles: responses at 15 min after pretreatments with SR-48968 and CP-99994. Data represent means ± SEM. See legend of Figure 3 for further explanation.

View larger version (20K): [in this window] [in a new window]   Figure 6.   Effect of pretreatments with CP-99994 and SR-48968 in combination on the peak responses of RL and Cdyn to cigarette smoke in Ova-sensitized guinea pigs. Open bars: baselines averaged over six consecutive breaths immediately before smoke inhalation; filled bars: first-phase responses averaged over six consecutive breaths with peak increase in RL that occurred within 20 breaths after smoke inhalation; hatched bars: second-phase responses averaged over breaths 75 to 80 after smoke inhalation. Data represent means ± SEM of 13 guinea pigs. *Significantly different from baseline; significant difference comparing corresponding data between before and after pretreatments with CP-99994 and SR-48968. See legend of Figure 4 for further explanation.

View larger version (23K): [in this window] [in a new window]   Figure 7.   Effect of pretreatments with CP-99994 and SR-48968 on the bronchomotor responses to ACh in Ova-sensitized guinea pigs. ACh (5.06 µg/kg, intravenously) was injected at the arrow. Open circles: responses to ACh during control; filled circles: responses at 15 min after pretreatments with CP-99994 and SR-48968 (45 min after the previous ACh challenge). Data represent means ± SEM of six animals. See legend of Figure 3 for further explanation.

Study Series 3: Assessments of Airway Inflammation and Tachykinin Release

The total number of leukocytes in the BALF collected from Ova-sensitized animals exceeded that of control animals by 73%, indicating that airway inflammation was induced by the Ova sensitization. In particular, the numbers of both eosinophils and neutrophils in the BALF of Ova-sensitized animals increased by > 250% (Table 2).

At the baseline (before CS) condition, there was no significant difference in either SP-LI or CGRP-LI in the BALF between control (n = 5) and Ova-sensitized (n = 5) animals (Figure 8). However, SP-LI was 6.98 ± 1.17 fmol/ml and CGRP-LI was 4.08 ± 0.31 fmol/ml in the BALF obtained from sensitized animals (n = 5) after the CS inhalation; these levels were significantly higher than those obtained from control animals after the CS inhalation (n = 5; SP-LI: 2.66 ± 0.41 fmol/ml, p < 0.05; CGRP-LI: 1.84 ± 0.27 fmol/ml, p < 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 8.   Effect of cigarette smoke inhalation challenge on CGRP-LI and SP-LI measured in the BALF of control (left panels) and Ova-sensitized animals (right panels). Open bars: baselines (before smoke inhalation challenge); filled bars: BAL obtained within 1 min after cigarette smoke inhalation challenge (15 ml, 100% concentration). Data represent means ± SEM of five guinea pigs in each subgroup. *Significantly different from baseline values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Results obtained from the present study show that Ova-sensitized guinea pigs develop airway hyperresponsiveness to nonallergic bronchoactive challenges, as evidenced by the elevated bronchomotor response to ACh (Figure 1). The exaggerated response is accompanied by the airway inflammatory reaction, as indicated by the marked increases in the numbers of eosinophils and neutrophils in the BALF (Table 2). Hence, our results are consistent with those reported by previous investigators using the same animal model (13, 16, 19, 22, 23). More importantly, our results further indicate that the acute bronchoconstrictive effect of inhaling a small amount of CS is also markedly augmented by Ova sensitization. We further conclude that the endogenous tachykinins play a primary role in this exaggerated response because the augmented effect was completely abolished by pretreatment of the animals with NK₁- and NK₂-receptor antagonists. The lack of response to CS after treatment with these antagonists was not due to tachyphylaxis because the responses of RL and Cdyn to repeated CS inhalation challenges were reproducible when more than 30 min elapsed between two challenges (Figure 3).

Recent studies reported from our laboratory have shown that inhalation of CS evokes acute bronchoconstriction in anesthetized guinea pigs (1, 2). The response consists of two distinct phases that are different in both time course and underlying mechanisms (2). The first phase is induced by activation of bronchopulmonary C-fiber afferents (1); the bronchoconstriction resulting from the cholinergic reflex and tachykinin release occurs rapidly, reaches a peak in 15 to 20 s, and then gradually declines toward the baseline. Subsequently, a relatively mild bronchoconstriction resulting from the action of arachidonic acid cyclooxygenase metabolite(s) slowly develops and reaches a plateau in 60 to 80 s. In the present study, inhalation of CS at a lower concentration (50%) did not evoke any significant initial response in control animals. However, in sharp contrast, the same CS inhalation challenge evoked an intense initial bronchoconstriction in the Ova-sensitized animals (Figures 2, 3, and 4).

Immunohistochemical studies have clearly demonstrated the localization of tachykinins and CGRP in the peripheral endings of the bronchopulmonary C-fiber afferents in various species, including humans (6, 24). These neuropeptides are known to have potent effects on airway smooth muscle tone, vascular permeability to protein, and airway mucus secretion (6). Tachykinins and CGRP have also been shown to interact with other cells in the lung (e.g., mast cells, leukocytes, epithelial cells) and to trigger the release of inflammatory mediators (25). Excitation of these sensory nerve endings by inhaled irritants such as CS may, therefore, lead to the "neurogenic inflammatory reaction" in the airways (6, 8, 28, 29).

Previous investigators have shown that the excitability of isolated C neurons in the guinea pig nodose ganglia that contain sensory neurons innervating the airways is enhanced by active sensitization with ovalbumin (14); it was suggested that the release of eicosanoids and histamine from the nodose ganglion may be involved. On the other hand, it has been well documented that a number of bronchoactive autacoids are released from various inflammatory cells in the allergen-sensitized airways (19, 30); some of these inflammatory mediators (e.g., histamine, prostaglandins) are known to enhance the sensitivity of pulmonary C-fiber endings (31). Hence, one plausible explanation for the present finding is that chronic airway inflammation that develops during the course of Ova sensitization may increase the excitability of tachykinin-containing C-fiber sensory endings in the lung (14, 31, 32). Thus, a given dose of CS may evoke a greater level of discharge from these endings and may cause larger amounts of neuropeptides to be released. Indeed, a substantial increase in the biosynthesis of tachykinins, measured by the messenger RNA levels, has been detected in the nodose ganglia of sensitized guinea pigs within 12 h after the Ova challenge (34). Moreover, our results obtained from RIA measurements of SP-LI and CGRP-LI in the BALF show that a significantly greater amount of these neuropeptides was released from the lung in response to CS inhalation challenge in sensitized guinea pigs than in control animals, despite the fact that no detectable difference was found between the two groups in the baseline levels of these neuropeptides (Figure 8).

To determine the relative roles of NK₁ and NK₂ receptors in mediating the augmented bronchoconstrictive responses to CS inhalation in sensitized guinea pigs, we selectively blocked these two subtypes of receptors in two separate groups of sensitized animals. Blocking the NK₂ receptors alone abolished more than 85% of the augmented response to CS, whereas blocking the NK₁ receptors alone did not significantly alter the response. This finding clearly indicates a more dominant role of NK₂ receptors.

Previous investigators have reported that reflex bronchoconstriction mediated through the cholinergic pathway plays a significant role in the allergen-induced airway hyperresponsiveness to inhaled bronchoactive agents in larger mammalian species (35). Indeed, it is known that stimulation of bronchopulmonary C-fiber afferents can elicit a cholinergic reflex bronchoconstriction (5). However, the cholinergic mechanism does not seem to be involved in the airway hyperresponsiveness to acute CS inhalation challenge induced by Ova sensitization in the present study, because the augmented response was not significantly altered by pretreatment with atropine. This discrepancy in the relative contributions of the cholinergic mechanism and endogenous tachykinins to Ova-induced airway hyperresponsiveness is probably related to species differences (6). In contrast to the response to inhaled CS, pretreatments with the NK-receptor antagonists did not significantly reduce the enhanced bronchomotor response to ACh injection (Figure 7). This disparity can be explained by the fact that ACh- induced bronchoconstriction is mediated primarily through the activation of M₃-receptors on the airway smooth muscles, and not the release of tachykinins from bronchopulmonary C fibers.

In conclusion, the results of this study show that Ova sensitization induces airway inflammation and bronchial hyperresponsiveness to acute CS inhalation challenge in anesthetized guinea pigs. Activation of the NK₂ receptor in the airway smooth muscle is primarily responsible for the augmented bronchoconstrictive response to CS inhalation, and an increased release of tachykinins in the lung is probably involved.