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Nov 15, 1999 - gas hyperpnea-induced bronchoconstriction in guinea pigs. J. Appl. Physiol. 1994; 76: 1150–5. 15 Von Neergaard K, Wirz K. Die Messung der.
Allergology International (2000) 49: 99–104

Original Article

Airway responsiveness to acetylcholine or capsaicin in immature and mature guinea pigs in vivo Hirokazu Arakawa,1,2 Hiroyuki Mochizuki,1 Kenichi Tokuyama,1 Akihiro Morikawa1 and Jan Lötvall3 1

Department of Pediatrics, Gunma University School of Medicine, Maebashi, Gunma, 2Saku Central Hospital, Unit of Pediatrics, Nagano, Japan and 3Department of Clinical Pharmacology, Sahrgrens Hospital, Göteborg University, Göteborg, Sweden

ABSTRACT Non-specific airway hyperresponsiveness is a characteristic feature of bronchial asthma. Airway reactivity to cholinergics may change with age during childhood. The present paper describes tests to see if the airway responsiveness induced by acetylcholine (ACh) or capsaicin, which causes a release of endogenous tachykinins from the sensory nerve, changes with age in guinea pigs. Changes in lung resistance (RL) were measured to monitor airflow obstruction caused by intravenous ACh or capsaicin in anesthetized immature guinea pigs (aged 2 weeks), and to compare with those of adult guinea pigs (aged 11 weeks). It was shown that both ACh and capsaicin induced a rapid and dose-dependent increase in RL both in immature and adult animals. The two age groups had similar responsiveness to ACh or capsaicin in terms of potency or sensitivity. The response induced by capsaicin was not affected by atropine. In addition, capsaicin caused no extravasation of Evans Blue dye as an index of airway plasma exudation in both age groups. The airflow obstruction induced by i.v. capsaicin is mainly due to a cholinergic-unrelated smooth muscle contraction. These results suggest that airway smooth muscle responsiveness to cholinergics or capsaicin is comparable in both immature and adult guinea pigs in vivo.

Correspondence: Hirokazu Arakawa MD PhD, Department of Pediatrics, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. Email: [email protected] Received 17 May 1999. Accepted for publication 15 November 1999.

Key words: aging, airway obstruction, asthma, capsaicin, neuropeptide.

INTRODUCTION Reversible airway constriction and prolonged nonspecific airway hyperresponsiveness (AHR) are characteristic features of asthma.1 The degree of AHR to cholinergics shows a good correlation with the severity of asthmatic symptoms.2 A few studies have ascertained how variable AHR is with growth in childhood.3,4 We have previously found that age-related changes of AHR to methacholine occur in asthmatic children and agematched controls from 2 to 13 years of age.5 However, we do not know the mechanism behind age-related changes in AHR from younger children to adults. Smooth muscle contraction, airway edema resulting from airway plasma exudation or mucus plug formation may contribute to airflow obstruction or AHR.6,7 Therefore, maturational changes in airway smooth muscle or microvasculature may be important for the age-related changes in AHR during childhood. In in vitro and in vivo animal experimental models, several studies have demonstrated the effects of maturation on airway reactivity to bronchoconstrictors, including chemical mediators and tachykinins.8–11 It has been found that airway tissues from immature guinea pigs, swine or humans exhibit a decrease in reactivity to tachykinins, such as substance P, neurokinin A or neurokinin B.10–13 In contrast, capsaicin, which stimulates the sensory nerves and releases endogenous tachykinins from the nerve endings, causes similar bronchoconstriction both in immature and adult guinea pigs.14

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The aim of the present study was to evaluate whether airway reactivities to neuromuscular agents, such as acetylcholine (ACh) or capsaicin, in immature guinea pigs differ from those in adults. To do this, we measured airflow obstruction and airway plasma exudation induced by these agents in both age groups. We also evaluated whether cholinergic reflexes are involved in capsaicininduced airflow obstruction in both age groups.

METHODS Animal preparation and measurement of lung resistance

Finally, eight immature and eight adult animals were intravenously administered with Evans Blue dye (20 mg/kg) over 1 min. A minute later, 10 µg/kg capsaicin or saline (n = 4 for each group) was injected intravenously. In all of these experiments, hyperinflation of the lungs, twice with the tidal volume by blocking the ventilatior outflow, was used between the ACh or capsaicin injections to avoid lung atelectasis. For each dose–response curve elicited by ACh or capsaicin, the estimated log dose that would have caused a 200% increase in RL in comparison with the post-saline baseline value (PD200) or that would have produced a half-maximal effect (ED50) was calculated by log-linear interpolation as an index of sensitivity or potency, respectively.

Experiments were performed using male Dunkin Hartley guinea pigs divided into two age groups: immature (199 ± 2 g, 2 weeks) and adult (505 ± 4 g, 11 weeks).8,9 The animal preparation and measurement of lung resistance (RL) were done as previously described. Briefly, guinea pigs were intramuscularly anesthetized with a 3:2 mixture of ketamine and xylazine (2 mL/kg). Each animal was mechanically ventilated through a tracheal cannula (10 mm in length and 2.7 mm in inner diameter in adults and 6 mm in length and 2.0 mm in inner diameter in immature animals) inserted into the lumen of the cervical trachea in a tracheostomy. A tidal volume of 10 mL/kg and a frequency of 60 breaths/min were used. Airflow was measured with a pneumotachograph connected to a transducer. Transpulmonary pressure was measured with a pressure transducer, 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. All signals were monitored with a 12-bit analog digital board connected to a Macintosh II computer. The RL was calculated according to the method developed by von Neergaard and Wirz.15 We pretreated all animals with suxamethonium (5 mg/kg i.v.) 10 min before the experiment to avoid artefacts induced by spontaneous breathing.

Six minutes after the administration of 10 µg/kg capsaicin, the animals were disconnected from the ventilator and the thoracic cavity was opened. A catheter was inserted into the aorta through the left ventricle. Each animal was perfused with 100 mL/kg of 0.9% NaCl to remove dye within the bronchial circulation. After perfusion through this route, another catheter was inserted into the pulmonary artery and the pulmonary circulation was then perfused with 50 mL/kg saline. The trachea and lungs were dissected out en bloc. Then, the parenchyma was scraped off carefully and the extraneous tissue removed. The trachea, main bronchi and intrapulmonary airways were separated from one another. All airway tissue was dried, using a freeze dryer, for 24 h. All the tissue was then weighed dry, and Evans Blue dye was extracted, in 2 mL of formamide, in a 40°C water bath for 24 h. Absorption at 620 nm was measured with a spectrophotometer. The extracted Evans Blue dye was quantitated by interpolation on a standard curve of dye concentrations in a range of 0–10 µg/mL, and expressed as ng/mg dry tissue.

Protocol

Drugs and chemicals

Acetylcholine dose–response curves from 0.3 to 100 µg/kg were elicited in immature and adult guinea pigs (n = 8 for each group). In a separate experiment, six immature and six adult animals received capsaicin. Capsaicin was administered intravenously in half-log incremental doses from 0.3 to 100 µg/kg. Additional animals from both age groups (n = 6 for each age group) were pretreated with atropine (1 mg/kg, i.v.) 10 min before capsaicin administration.

The drugs and chemicals used were: ketamine hydrochloride (Park-Davis SA, Barcelona, Spain), xylazine chloride (Bayer Sverige AB, Göteborg, Sweden), suxamethonium chloride (KabiVitum AB, Stockholm, Sweden), Evans Blue dye (Aldrich Chemical Co., Milwaukee, WI, USA), ACh, capsaicin (Sigma Chemical Co., St Louis, MO, USA). Capsaicin was initially dissolved in an ethanol (0.2 mL), Tween 80 (0.2 mL) and saline (9.6 mL) mixture to 1 mg/mL and serially diluted with saline.

Detection of extravasation of Evans Blue dye

AIRWAY RESPONSIVENESS AND MATURATION

Data analysis The data were reported as the mean ± SEM. Nonparametric analysis, the Mann–Whitney U-test, was used to analyze a significant difference, if any. A P value less than 0.05 was considered significant. The data were analyzed with a Macintosh computer, using a standard statistical package (STATVIEW II; Abacus Concepts Inc., Berkeley, CA, USA).

RESULTS Baseline RL was significantly higher in immature animals than in adults (Table 1, Fig. 1). The time course of RL induced by 10 µg/kg of ACh or capsaicin is shown in Fig. 1. The time to the peak response after either ACh or capsaicin within a 6 min observation period was approximately 15–30 s (Fig. 2). In both immature and adult animals, intravenously administered ACh or capsaicin induced a dose-related Table 1 Bodyweight, baseline lung resistance and mean systemic blood pressure in immature and adult guinea pigs Immature Age (weeks) Bodyweight (g) Lung resistance (cmH2O/mL per s) Blood pressure (mmHg)

increase in RL in both age groups (Fig. 1a–c). Peak RL after ACh or capsaicin was significantly higher in immature animals than in adults at every dose studied. However, a half-maximal effect after ACh in immature animals did not differ from that of adult animals (ED50 13.5 ± 3.2 and 9.2 ± 2.8 µg/kg for immature and adults, respectively, P = 0.47). The sensitivity of immature guinea pigs to ACh was similar to that of adult animals (PD200 2.9 ± 0.5 and 3.0 ± 0.2 µg/kg, respectively; P = 0.85). There were no significant differences in ED50 and PD200 in responsiveness to capsaicin between the two age groups (ED50 10.0 ± 2.5 and 7.4 ± 1.9 µg/kg for immature and adults, respectively, P = 0.71; PD200 2.6 ± 0.6 and 1.8 ± 0.4 µg/kg, respectively, P = 0.32). Pretreatment with atropine (1 mg/kg) did not affect the dose–response curves obtained by intravenous administration of capsaicin in either age group (Fig. 1b,c). At all airway levels, except in intrapulmonary airways in adults, no significant difference was found in the extravasation of Evans Blue dye after 10 µg/kg capsaicin compared with that after saline both in immature and adult animals (Table 2).

Adult

2 199 ± 2

11 505 ± 4

0.39 ± 0.03* 35 ± 4*

0.22 ± 0.02 49 ± 4

Values are the mean ± SEM. *P < 0.01.

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DISCUSSION We have shown that immature guinea pigs exhibit similar bronchial reactivity to acetylcholine (ACh) or capsaicin as that of adult animals, when both sensitivity and potency to these agents are compared. An immediate increase in

Fig. 1 Dose–response curves obtained by intravenous administration of acetylcholine (a) or capsaicin (b) in adult (m) and immature (d) guinea pigs. (c) Effects of atropine on capsaicin dose-response curves in adult (n) and immature (s) animals. Peak lung resistance after acetylcholine or capsaicin treatment was significantly higher in immature animals than in adults at every dose studied.

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H ARAKAWA ET AL. Fig. 2 Time course of lung resistance induced by acetylcholine (a) or capsaicin (b) in immature (d) and adult (n) guinea pigs.

Table 2 Effects of maturation on capsaicin-induced compared with saline-induced airway plasma exudation in guinea pigs Immature Number Trachea Main bronchi IPA

Adult

Saline

Capsaicin

Saline

Capsaicin

4 45 ± 7 50 ± 10 56 ± 7

4 40 ± 13 73 ± 16 67 ± 13

4 68 ± 7 51 ± 10 54 ± 8

4 57 ± 5 77 ± 18 67 ± 7*

Airway plasma exudation after capsaicin (10 µg/kg) or saline (0.9% NaCl) induction was quantitated by measurement of extravasation of Evans Blue dye (ng/mg dry tissue). Values are the mean ± SEM. *P < 0.05 compared with animals of corresponding ages given saline. IPA, intrapulmonary airways.

responsiveness to ACh or capsaicin was found within a minute. Capsaicin-induced airflow obstruction was not altered by pretreatment with atropine in both age groups. In addition, capsaicin caused no airway plasma exudation, as is the case with cholinergics,7 indicating that cholinergic-unrelated smooth muscle contractions play a major role in capsaicin-induced airflow obstruction. We conclude that AHR to cholinergics or capsaicin is comparable both in immature and adult guinea pigs, probably due to the similar responsiveness of airway smooth muscle in the two age groups. We have previously found age-related changes in AHR to methacholine both in asthmatic children and agematched controls from 2 to 13 years of age.5 In both groups of children aged 2–7 years, the threshold point of respiratory resistance or the inflection point of transcutaneous oxygen pressure decreased, while after age 8 the values gradually increased. Interestingly, AHR in the

younger children (aged 2–3 years) was almost similar to those of older children (aged 12–13 years).5 However, we did not identify the mechanisms behind age-related changes in AHR to cholinergics in childhood. Smooth muscle contraction, airway edema or mucus plug formation may contribute to the airflow obstruction or AHR.6,7 Therefore, we studied which component plays a major role in age-related changes in the airflow obstruction induced by ACh or capsaicin, by using an animal model. In the present study, both immature and adult animals had similar responsiveness to ACh. Both an immediate increase in RL in the present study and no airway plasma exudation in the response to ACh, as previously reported,7 indicate that smooth muscle contraction plays a major role in ACh-induced airflow obstruction. Thus, our result suggests that immature and adult animals have similar responsiveness of the airway smooth muscle to ACh.

AIRWAY RESPONSIVENESS AND MATURATION

We have previously found that exogenously administered tachykinins, such as substance P and neurokinin A, induce less airflow obstruction and airway plasma exudation in immature guinea pigs than in adults.10 Therefore, we hypothesized that tachykinins released endogenously from sensory nerve endings by i.v. capsaicin may be less potent in causing airflow obstruction in immature animals. Contrary to our expectation, in the present study immature guinea pigs exhibited similar bronchial reactivity to capsaicin compared with adult animals, when both sensitivity and potency to capsaicin were compared. Our results further expand the study of Murphy and coworkers,14 which showed that immature guinea pigs have reactivity to capsaicin similar to adult guinea pigs, although they used the sensitivity as an index. In addition, they ventilated the animals with humidified 100% O2, while we used room air. There may be some possible explanations for the discrepancy, in our previous study, of lower responsiveness to exogenously applied tachykinins in immature animals. The involvement of cholinergic reflexes in response to i.v. capsaicin is still controversial.16–18 In addition, it remains unclear whether there is an agerelated change in the involvement of cholinergic reflexes to capsaicin. Therefore, we studied the effect of atropine on the capsaicin-induced airway response both in immature and adult guinea pigs. In the present study, bronchoconstrictor response to capsaicin was unaffected by atropine in the two age groups. In our preliminary study, the dose of atropine we used completely abolished the bronchoconstriction induced by i.v. ACh. Our result suggests that similar responsiveness to capsaicin in the two age groups cannot be attributed to the cholinergic reflex. In addition, we also considered that the airway plasma exudation induced by capsaicin could show age-related differences between the exogenous tachykinins and capsaicin, because airway plasma exudation partly contributes to airflow obstruction induced by several mediators.6,7 In the present study, 10 µg/kg capsaicin induced little airway plasma exudation in immature animals as well as adults. Murai and coworkers have found that 100 µg/kg capsaicin induces airway plasma exudation and that pretreatment with FK224, NK1 and NK2 receptor antagonists inhibits this response.19 We did not choose this dose of capsaicin, because an almost maximal increase in RL was already seen with 10 µg/kg capsaicin in our model. Thus, our result suggests that smooth muscle contractions play a major role in

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capsaicin-induced airflow obstruction, at least up to 10 µg/kg capsaicin. Finally, another possible explanation is that the amount of tachykinin in the airway nerve endings decreases with age.20 In addition, capsaicin-induced airflow obstruction could partly involve the release of other mediators from sources other than sensory nerves, and airway responsiveness to these mediators may change with age. Further studies will be necessary to clarify these points. In conclusion, smooth muscle responsiveness to cholinergics or capsaicin may develop well in immature airways as well as in adults, but further studies in man are required to answer this question.

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18 Buchan P, Adocock JJ. Capsaicin-induced bronchoconstriction in the guinea-pig: Contribution of vagal cholinergic reflexes, local axon reflexes and their modulation by BW443C81. Br. J. Pharmacol. 1992; 105: 448–52. 19 Murai M, Morimoto H, Maeda Y, Kiyotoh S, Nishikawa M, Fujii T. Effects of FK224, a novel compound NK1 and NK2 receptor antagonist, on airway constriction and airway edema induced by neurokinins and sensory nerve stimulation in guinea pigs. J. Pharmacol. Exp. Ther. 1992; 262: 403–8. 20 Asai H, Yoshihara S, Ichimura T, Iguchi K, Yanaihara N. Changes in substance P and vasoactive intestinal polypeptide levels in the respiratory tract of aging guinea pigs. Biomed. Res. 1991; 12: 41–6.