Journal of Applied Physiology AJP: Lung Cellular and Molecular Physiology
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J Appl Physiol 105: 187-196, 2008. First published April 17, 2008; doi:10.1152/japplphysiol.01048.2007
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Adenosine induces a cholinergic tracheal reflex contraction in guinea pigs in vivo via an adenosine A1 receptor-dependent mechanism

Sandra M. Reynolds,1 Reginald Docherty,2 Jon Robbins,2 Domenico Spina,1 and Clive P. Page1

1Pharmaceutical Science Research Division, The Sackler Institute of Pulmonary Pharmacology, School of Biomedical and Health Science, King's College London, and 2Wolfson Centre for Age-Related Research, School of Biomedical and Health Science, King's College London, London, United Kingdom

Submitted 1 October 2007 ; accepted in final form 10 April 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenosine induces dyspnea, cough, and airways obstruction in asthma, a phenomenon that also occurs in various sensitized animal models in which a neuronal involvement has been implicated. Although adenosine has been suggested to activate cholinergic nerves, the precise mechanism has not been established. In the present study, the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) induced a cholinergic reflex, causing tracheal smooth muscle contraction that was significantly inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 100 µg/kg) (P < 0.05) in anesthetized animals. Furthermore, the adenosine A2 agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) induced a small reflex, whereas the A3 selective agonist N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) was without effect. The tracheal reflex induced by CPA was also inhibited by recurrent nerve ligation or muscarinic receptor blockade (P < 0.001), indicating that a cholinergic neuronal mechanism of action accounted for this response. The cholinergic reflex in response to aerosolized CPA was significantly greater in passively sensitized compared with naive guinea pigs (P < 0.01). Chronic capsaicin treatment, which inhibited sensory nerve function, failed to inhibit CPA-induced reflex tracheal contractions in passively sensitized guinea pigs, although the local anesthetic lidocaine inhibited CPA-induced tracheal contractions. The effects of CPA on the reflex response was not dependent on the release of histamine from tissue mast cells or endogenous prostaglandins as shown by the lack of effect of the histamine H1 receptor antagonist pyrilamine (1 mg/kg) or the cyclooxygenase inhibitor meclofenamic acid (3 mg/kg), respectively. In conclusion, activation of pulmonary adenosine A1 receptors can stimulate cholinergic reflexes, and these reflexes are increased in allergic guinea pigs.

N6-cyclopentyladenosine; cholinergic reflex; adenosine 5'-monophosphate; capsaicin; passively sensitized

AIRWAY HYPERRESPONSIVENESS (AHR) to a variety of bronchoconstrictor stimuli is a characteristic feature of subjects with asthma. Adenosine and related purines, like adenosine 5'-monophosphate (AMP), are known to induce airways obstruction in asthmatic subjects, whereas they have no effect in normal subjects (42). The implications of this clinical finding are significant because AMP could clearly be a phenotypic marker of AHR, a characteristic feature of asthma and, possibly, a better marker of this phenomenon compared with methacholine or histamine, which although widely used, can induce bronchoconstriction in both healthy subjects and in subjects with asthma. However, the precise mechanism of adenosine-induced airways obstruction is still undetermined, although a number of possible mechanisms have been proposed, including a role for mast cell-derived mediators (34) and activation of neuronal pathways (24, 38).

The activation of airway nerves by adenosine in mediating pulmonary responses may also be inferred from several clinical observations. In humans, adenosine induces dyspnea, cough, and irritation of the throat (2). The sensation of dyspnea is associated with asthma symptoms, and it is thought to be brought about by irregular vagal sensory nerve activity. The intravenous administration of adenosine also caused dyspnea in normal subjects in the absence of bronchoconstriction, but the time latency between the dyspneic response and the ventilatory and heart rate response ruled out the involvement of peripheral chemoreceptor activation or brain stem respiratory stimulation, suggesting that it is likely due to secondary stimulation of receptors in the lung, probably C fibers (5). Furthermore, inhaled ipratropium bromide, a muscarinic receptor antagonist, significantly attenuated the bronchoconstrictor effect to AMP in subjects with asthma (35). The possible contribution of mast cells via an adenosine A2B receptor-dependent mechanism (19) and the contribution of airway smooth muscle adenosine A1 receptors to this response cannot be ignored, because bronchial smooth muscle from asthmatic, but not healthy, subjects contracted to adenosine via an adenosine A1 receptor-dependent mechanism (3), and adenosine A1 receptor expression is upregulated in bronchial tissue obtained from subjects with asthma (4). Hence, airway responses to adenosine in humans appear to be complex and may involve different cell types and activation of different adenosine receptor subtypes.

A number of studies using experimental animals have also implicated a role for adenosine A1 receptors in mediating airways obstruction to adenosine. For example, the adenosine A1-receptor agonist CPA selectively induces airway obstruction only in sensitized guinea pigs (24) and rabbits (1, 13, 33). However, atypical (18) and adenosine A1, A2B, and A3 receptors (14, 21) have been suggested to mediate airways obstruction in response to adenosine in the Brown-Norway (BN) rat and mice, respectively, underlying important species and strain differences. Furthermore, adenosine has been shown to activate pulmonary C fibers in anesthetized rats via an adenosine A1 receptor-dependent mechanism (20), and the adenosine A1 receptor agonist CPA has been demonstrated to activate nodose, but not jugular ganglion-derived, C fibers (10). Our laboratory has previously shown that adenosine may induce airways obstruction in vivo via activation of airway sensory nerves that partly involved a cholinergic reflex (24). The aim of the present study was to further investigate the mechanisms whereby adenosine receptor agonists can activate airway reflexes in anesthetized, naive, and passively sensitized guinea pigs and in particular to investigate whether adenosine receptor agonists could initiate a cholinergic reflex leading to tracheal smooth muscle contraction, because our laboratory's previous work (24) had suggested a role for neuronal mechanisms in adenosine-induced airways obstruction.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.   Male Dunkin-Hartley guinea pigs (300–450 g) supplied by Harlan UK were used throughout this study. Guinea pigs were housed in the King's College London biological services unit and given free access to food and water. All experiments described were undertaken under the Animals (Scientific Procedures) Act (1986).

Measurement of lung function.   Guinea pigs were anesthetized with urethane (1.5 g/kg ip) and placed ventral side up on a heating pad. This dose of urethane produces a deep anesthesia lasting up to 10 h, although the present experiments described in this study rarely lasted longer than 4 h. The adequacy of anesthesia was assessed throughout the course of the experiment by checking for reflex limb movement using the pinch reflex of the foot. Blood pressure and heart rate were also continuously monitored. Guinea pigs were ventilated (60 breaths/min, 6 ml/kg) via a tracheal cannula (inserted through cartilage rings 15–16).

Changes in total lung resistance (RL) were measured using an automated lung function recording system (Pulmonary Monitoring system 6.0, Mumed, London, UK) and displayed in real time on a personal computer. The flow signal was obtained by connecting the tracheal cannula to a pneumotachograph and a pressure transducer (±2 cmH2O; Validyne Engineering, Northridge, CA). Flow was integrated to give a measure of tidal volume. An intrathoracic cannula was inserted between the 10th and 11th intercostal space and connected to the negative side of a pressure transducer (±20 cmH2O, Validyne Engineering). The positive side of the transducer was connected to a side port of the pneumotachograph proximal to the animal for the determination of transpulmonary pressure.

Succinylcholine chloride (2 mg/kg) was administered by subcutaneous injection to induce paralysis to eliminate any spontaneous breathing, which would interfere with the sensitive measurement of tracheal tension in all animals. Adequacy of anesthesia was determined by continuous monitoring of heart rate and blood pressure.

Measurement of tracheal tension.   Two stainless steel fishhooks were positioned between two to three cartilage rings (rings 5–7) caudal to the larynx on the lateral aspect of the trachea and rostral to the tracheal cannula (Fig. 1). The hooks are placed directly opposite each other on the most lateral side of the trachea, and care was taken not to damage the recurrent laryngeal nerves. The hooks were placed in such a manner that they were directly opposite each other. Care was also taken to ensure that the entry and exit points of the hook was in the same plane. A length of silk braided suture (4-0) was tied to the hooks, one attached to a fixed bar and the other to the force transducer with its output displayed on a MacLab. A small slit was made on the ventral side of the trachea just caudal to the hooks to allow oxygenated warmed (37°C) Krebs-Henseleit buffer [KH; composition (in mM): 118 NaCl, 5.4 KCl, 1 NaHPO4, 1.2 MgSO4, 1.9 CaCl2 25 NaHCO3, and 11.1 glucose] to be perfused (2 ml/min) through the trachea. A short length (10 cm) of PE-60 tubing, frayed at its distal end to prevent occlusion, was inserted through the slit and through the larynx until it exited out the nasal passage and was connected to a gentle vacuum to remove the KH perfusate. The frayed end was situated in the most rostral portion of the trachea, making sure it did not pass through the larynx. Care was also taken not to damage the epithelium when inserting the cannula by maneuvering the cannula gently along the airway. Baseline tension was set at 1.5–2 g as previously described (25).


Figure 1
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  		Fig. 1. Diagrammatic representation of the vagal innervation of the extrapulmonary and intrapulmonary airways of the guinea pig. Parasympathetic nerves within the recurrent laryngeal nerves (RLN) innervate the tracheal wall. The upper trachea was perfused with Krebs-Henseleit (KH) solution containing indomethacin (3 µM), phentolamine (1 µM), and propranolol (2 µM) to rule out the effect of endogenously release prostanoids and catecholamines on airway smooth muscle function and permitted the measurement of tracheal smooth muscle tension in response to drugs applied topically to the airways via a nebulizer or following intravenous administration of substances. The addition of selective receptor antagonists to the KH solution prevented the direct stimulation of tracheal airway smooth muscle from systemically administered bronchoconstrictor agents. Total lung resistance (RL) was also used to monitor airways obstruction in the intrapulmonary airways following intravenous or inhaled administration of drug. Ligation of the RLNs (*) or administration of atropine into the KH solution inhibited contraction of tracheal smooth muscle in response to reflex activation of parasympathetic nerves. NG, nodose ganglion; JG, jugular ganglion; CNS, central nervous system.

 
In all experiments carried out in this study, KH solution contained indomethacin (3 µM), propranolol (2 µM), and phentolamine (1 µM) (6). These drugs were used to block any action of endogenously released prostaglandins or neuronal-released catecholamines on tracheal muscle tension. Similarly, pyrilamine (1 µM; histamine H1 antagonist), S-4-[2-bis(cyclohexylamino)methyleneamino]-3-(2-naphthalenyl)-1-oxopropylamino]benzyl tributyl phosphonium chloride hydrochloride (WIN-64338; 1 µM; bradykinin B2 antagonist) (6), and 8-cyclopentyl-1,3-dipropylxanthine; (DPCPX; 1 µM; adenosine A1 receptor antagonist) were added to the KH perfusate in all experiments to block any direct effect of the autacoids used to elicit tracheal smooth muscle contraction. An equilibration period of 20 min was allowed to elapse to establish baselines for tracheal tension and RL. Atropine (1 µM) was added to the KH solution at the end of each experiment to determine whether the reflex was cholinergic in nature.

The abdominal aorta and vena cava were cannulated to monitor arterial blood pressure and for the delivery of drugs, respectively. This method of delivery was chosen to prevent disruption to blood flow to the trachea and surrounding nerves. Heparinized saline (100 U/ml) was used to deliver drugs and prevent obstruction of the cannula due to the formation of blood clots.

Passively sensitization protocol.   Guinea pigs were immunized by intraperitoneal injection of 2.5 mg/ml ovalbumin in saline (0.1 ml) plus aluminium hydroxide (0.9 ml), and after 10 days this procedure was repeated. Each animal received 5 mg/kg ovalbumin in total. After a further 7 days, blood was collected in heparin (0.2 ml; 100 U/ml) by cardiac puncture under anesthesia with urethane (1.75 g/kg ip). Blood was centrifuged (2,000 g for 15 min) and the plasma was removed and stored (–20°C). For passive sensitization, guinea pig anti-ovalbumin plasma was injected intravenously into the foot vein of naïve recipient guinea pigs (1 ml/animal). Lung function was recorded 7–10 days later. Our laboratory has previously used passive immunization to investigate airways obstruction following activation of adenosine A1 receptors (24) to model the clinical observation that adenosine A1 receptors mediate contraction of human isolated bronchial tissue (3) and our laboratory's recent observation of increased expression of this receptor in biopsies from mildly asthmatic subjects (4). In all experiments, ovalbumin (100 µg/kg) was administered intravenously at the end of each experiment to confirm a successful sensitization in passively sensitised guinea pigs.

Capsaicin treatment.   In some experiments, guinea pigs were chronically treated with capsaicin, before passive sensitization as previously described (24). Briefly, naive guinea pigs were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). Pyrilamine (10 mg/kg) and salbutamol (1 mg/kg) were administered to protect animals from airways obstruction induced by the acute administration of capsaicin. Fifteen minutes later, animals were subcutaneously injected with capsaicin or vehicle (ethanol-Tween 80–0.9% saline; 1:1:8) in the dorsal region of the neck. Animals were treated twice a day (6 h apart) on 3 consecutive days to increasing doses of capsaicin, totaling 80 mg/kg.

Intravenous studies.   Dose-response curves to intravenous administration of histamine (1, 4 µg/kg) were performed in naive guinea pigs after stable recordings of RL, tracheal tension, and blood pressure were established. The different doses of histamine were administered at 3-min intervals after RL, tracheal tension, and blood pressure had returned to baseline. Bradykinin (1, 2 µg/kg) and N6-cyclopentyladenosine (CPA; 1, 2 µg/kg) dose-response curves were performed 10 min after the completion of the dose-response curves to histamine. In some experiments, the response to CPA (2 µg/kg) was also evaluated following intravenous administration of DPCPX (100 µg/kg).

Nebulized studies.   Anesthetized animals were exposed to saline for the purpose of obtaining a baseline response and exposed 2 min later with CPA (10 mg/ml), nebulized for 10 s. Similarly, AMP (10 mg/ml) or 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-2160; 10 mg/ml) was then nebulized for a period of 10 s. The different spasmogens (AMP, CPA, CGS-21680) were administered at 5-min intervals or when the change in RL, tracheal tension and blood pressure had returned to baseline. The spasmogens were randomized in order of administration. In all experiments, atropine (1 µM) was administered to the tracheal perfusate and tracheal tension increased in response to nebulized CPA or AMP or CGS-2160 (10 mg/ml). In passively sensitized animals, ovalbumin (100 µg/kg) was also administered intravenously to demonstrate that the animals were sensitized.

Recurrent laryngeal nerve ligation studies.   The vagus nerve innervates the trachea via the recurrent laryngeal nerves (RLNs) and severing these nerves on either side below the tracheal segment (tracheal rings 10–11) deprives that area of the trachea of all input from preganglionic parasympathetic fibers (Fig. 1). Tracheal reflexes in response to CPA (10 mg/ml) were measured before and 10 min after RLN ligation. RLN ligation was confirmed at autopsy.

Pharmacological studies.   Guinea pigs were chronically treated with capsaicin as previously described (24) and then passively sensitized with the anti-ovalbumin plasma 4 days after the last injection of the capsaicin and used in experiments 7 days later. All animals were challenged with CPA (10 mg/ml) for 10 s at 10-min intervals or when the change in RL, tracheal tension, and blood pressure had returned to baseline. Similarly, AMP, bradykinin (10 mg/ml;10 s), and capsaicin (100 µg/kg) were also administered at 10 min intervals. Atropine (1 µM) was administered to the tracheal perfusate. Ovalbumin (100 µg/kg) was administered at the end of all experiments in passively sensitized guinea pigs.

The selective H1 antagonist pyrilamine (1 mg/kg) was administered in both naive and passively sensitized guinea pigs to assess the role of histamine release from mast cells in the CPA-induced cholinergic reflex. All animals received a histamine (1 mg/ml) challenge for 10 s followed by CPA (10 mg/ml) challenge. Spasmogens were administered at 5-min intervals or when the change in RL, tracheal tension, and blood pressure had returned to baseline. Pyrilamine was then administered, and 10 min later, histamine and CPA challenges were repeated. Atropine (1 µM) was administered afterward in the tracheal perfusate. Ovalbumin (100 µg/kg) was administered at the end of all experiments using passively sensitized guinea pigs.

In other experiments, meclofenamic acid (cyclooxygenase inhibitor; 3 mg/kg) was administered intravenously to assess the role of circulating or neural release of prostanoids on reflex induced-tracheal contractions and RL to nebulized CPA (10 mg/ml), which was administered both before and 15 min after meclofenamic acid (3 mg/ml).

The adenosine A1 antagonist DPCPX (100 µg/kg) was administered intravenously to assess whether the reflex initiated by CPA was adenosine A1 receptor-dependent. CPA (2 µg/kg) dose was chosen because it reliably produced tracheal cholinergic reflexes. Tracheal responses to CPA (2 µg/kg) were measured before and after 15 min DPCPX.

To further assess the role of nerves in CPA-induced cholinergic reflexes, the local anesthetic lidocaine was employed. In passively sensitised guinea pigs, bradykinin (1 mg/ml for 10 s) was nebulized, and this was followed 5 min later by CPA (10 mg/ml for 10 s) challenge in passively sensitised guinea pigs. Lidocaine (10 mg/ml) was then nebulized for 2 min. CPA and bradykinin challenges were then repeated.

In vitro contractility studies.   Male albino guinea pigs (250–350g) were killed by cervical dislocation, and the lungs were removed and placed in KH solution containing indomethacin (5 µM) and propranolol (1 µM). The trachea was isolated and 2-mm bronchial rings were sectioned and suspended between wire hooks, under 1 g of tension in 10-ml organ baths in oxygenated (95% O2-5% CO2) KH buffer at 37°C. Changes in tension were measured isometrically using a FT03C transducer and recorded using Maclab (version 3.3.8). Tissues were equilibrated for 45 min with changes in KH made every 10 min. Tissues were exposed to methacholine (10 µM) to establish sensitivity. After the contractile response had reached plateau, the tissues were washed at 10-min intervals and allowed to equilibrate for a further 30 min. Cumulative concentration response curves to CPA, AMP, CGS-21680, and 1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-b-D-ribofuranuronamide (IB-MECA; 0.3–10 µM) were performed to establish their effect on tracheal tension. In passively sensitized tissue, ovalbumin (10 µM) was added to all organ baths to establish whether indeed the tissue was sensitized.

Transfection method.   To establish whether adenosine ligands could interact with transient receptor potential vanilloid type 1 (TRPV1) directly, COS7 cells were grown in 50 ml flasks in DMEM culture medium (Sigma) supplemented with 10% fetal bovine serum, 0.1 mg/ml streptomycin, and 100 U/ml penicillin and then plated on to 22-mm glass coverslips that had been coated previously with polyornithine. Cells on coverslips were cultured in DMEM without FBS or antibiotics for ~90 min and then transfected with rat TRPV1 cDNA using lipofectamine (LF2000, Invitrogen) according to the manufacturer's instructions. Rat TRPV1 cDNA was a generous gift from Prof. David Julius (San Francisco, CA) to Dr R. Docherty. Transfected cells were then used for experiments 24–48 h following the transfection procedure.

Intracellular Ca2+ measurements.   COS7 cells were incubated in 5 µM indo-1 AM (Calbiochem) in the dark at 37°C for 45–60 min. The coverslips were removed from the Petri dishes and placed in a custom-built chamber that was superfused by a gravity-fed system at 10–12 ml/min at room temperature (21–23°C) on the stage of an epifluorescent inverted microscope (Nikon Diaphot 200). The superfusate consisted of (in mM) 130 NaCl, 11 glucose, 5 HEPES, 1 MgCl2, 1 CaCl2, and 3 KCl, pH 7.4 with NaOH (1 M).The indo dye was excited at 360 nm via a x40 fluoroobjective, and two emission wavelengths (405 and 488 nm) were measured simultaneously on a pair of photomultiplier tubes (Thorn EMI). The output voltages were relayed to a "ratio amplifier" (custom built by T. Dyett, University College, London, UK) and three signals (the readings at 405 nm and 488 nm, and the 405/488 ratio) were viewed and recorded using pClamp6 software (Axon Instruments). Data were stored on an optical disk (model LF-7010E, Panasonic) for further analysis.

Initially, an area of the coverslip with no cells present was used to offset background light levels, and this was routinely checked and adjusted between cells and dishes. A cell was placed in the center of a 25-µm-diameter circular field and the excitation intensity adjusted using neutral density filters to ensure minimal bleaching of the dye over 8 min. Furthermore, the excitation light pathway was interrupted when measurements were not being taken, again to minimize dye bleaching. Background light levels were subtracted from the individual photomultiplier measurements on line. A further test to check autofluoresence levels of the cells was to quench the dye with MnCl2 (2 mM) after the cells had been permeablized with ionomycin (10 µM). Autofluoresence was <1% of the unquenched cells.

Each measured Ca2+ ratio (R) was converted to approximate intracellular Ca2+ concentration ([Ca2+]i) using the equation [Ca2+]i = Kdapp [(R – Rmin)/(Rmax – R)] (16). Rmin was assessed by loading the cells with the dye as normal and then superfusing them in a bath solution with zero Ca2+ (i.e., Ca2+ omitted and 1 mM EGTA added, with a supplement of 250 µM BAPTA-AM) for 1 h. The ratio (Rmin) under these conditions was 0.22 (n = 20), and Rmax was assessed by placing the cells in a high (20 mM) Ca2+ solution and adding ionomycin (10 µM). Under these conditions, the ratio (Rmax) was 4.50 (n = 20). Kdapp was the apparent dissociation constant defined as the product of the listed constant for indo-1 AM (230 nM) (39) and the dynamic range of the 488-nm photomultiplier output (often termed β). The value of Kdapp was 722 nM.

Drugs.   The following reagents were used: Chicken egg ovalbumin (Sigma, UK), urethane (Sigma), CPA (Tocris, UK), AMP (Sigma), histamine (Sigma), bradykinin (Sigma), DPCPX (Tocris), capsaicin (Sigma), ketamine, xylazine, Tween, pyrilamine (Sigma),IB-MECA (Tocris), CGS-21680, WIN64338 (Tocris), DMSO (Sigma), ethanol (Sigma), aluminium hydroxide solution (Sanofi, Rio de Janeiro, Brazil), Lidocaine (Sigma), and meclofenamic acid (Sigma).

Statistical analysis.   Peak tracheal tension was calculated as an increase (in grams) above baseline. In vitro drug responses were expressed as percentage of maximal contraction to methacholine. In vivo drug responses are expressed as a percentage increase over baseline measurement of peak RL and expressed as arithmetic means ± SE. Paired Student's t-test was used to analyze differences between means. In some cases, one-way ANOVA was used to analyze treatment effects and differences in means compared with an appropriate multiple comparison test. Results were considered statistically significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Tracheal reflex: naive animals.   Intravenous administration of histamine and bradykinin induced a tracheal reflex contraction in naive guinea pigs consistent with previous published findings (6) (Figs. 2 and 3). Similarly, intravenous administration of the adenosine A1-selective agonist, CPA, also induced contraction of tracheal smooth muscle that was not a consequence of direct activation of airway smooth muscle, because the KH solution perfused through the trachea contained the adenosine A1 antagonist DPCPX (Fig. 3). Furthermore, tracheal reflexes in response to CPA (2 µg/kg) were significantly inhibited in animals treated with DPCPX (100 µg/kg; P < 0.05, Fig. 4). In control experiments, vehicle did not alter tracheal tone or baseline RL (data not shown). Furthermore, reproducible reflex responses were obtained in response to CPA (2 µg/kg) after a 10-min interval (0.20 ± 0.04 vs. 0.21 ± 0.03 g; n = 6; P > 0.05). This response was mediated by reflex activation of the parasympathetic nervous system, because tracheal reflexes to bradykinin and CPA were abolished following local administration of atropine in the perfusate (Fig. 3).


Figure 2
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  		Fig. 2. Reflex activation of tracheal smooth muscle in response to bradykinin, histamine, and CPA. Increase in tracheal tension (A) and baseline RL (B) in response to intravenously administered histamine ({circ}), bradykinin (bullet), and CPA ({square}) is shown. Vertical lines represent means ± SE; n = 4–6 animals per group.

 

Figure 3
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  		Fig. 3. Representative trace of the functional response to bradykinin and CPA. Increase in grams tension induced by intravenously administered bradykinin (1 µg/kg) and CPA (2 µg/kg) in the absence (A) and following (B and C) administration of atropine (1 µM) into the tracheal perfusate of an anesthetized guinea pig is shown.

 

Figure 4
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  		Fig. 4. Reflex activation of tracheal smooth muscle in response to CPA is mediated by adenosine A1 receptors. Increase in grams of tension induced by reflex tracheal smooth muscle contraction in response to intravenously administered CPA (2 µg/kg) in the absence (filled bar) and presence (gray bar) of intravenously administered DPCPX (100 µg/kg) in naive guinea pigs. Vertical lines represent means ± SE, n = 4 animals per group. *P < 0.05 compared with CPA only. Intravenous administration of vehicle control (10% DMSO and 90% saline) did not alter tracheal tone (open bar), n = 3 animals per group.

 
To further examine the nature of the tracheal reflex response, we investigated the effect of direct administration of CPA to the lung. Aerosolized CPA also induced a reflex tracheal contractile response that was attenuated by sectioning of the recurrent laryngeal nerves (Fig. 5A). This response to aersolized CPA was also attenuated by atropine (tracheal tension: 0.0012 ± 0.0005 g, n = 5 compared with control in Fig. 5A). There was no significant change in bronchoconstrictor response to CPA (Fig. 5B). The role of histamine release from mast cells in CPA-induced cholinergic reflexes experiments was investigated in naive guinea pigs. The intravenous administration of the H1 antagonist pyrilamine (1 mg/kg) inhibited aerosolized histamine but not CPA-induced cholinergic reflexes (Fig. 6) and is consistent with previous findings that demonstrated that pyrilamine inhibited histamine induced tracheal reflexes (6). When administered topically to the lung, histamine and not CPA caused airway obstruction that was sensitive to pyrilamine, consistent with the activation of airway smooth muscle histamine H1 receptors in the intrapulmonary airways (Fig. 6).


Figure 5
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  		Fig. 5. Innervation of tracheal smooth muscle is activated by motor input via the RLNs. Increase in grams of tension induced by reflex tracheal smooth muscle contraction (A) and % increase in RL (B) in response to nebulized CPA (10 mg/ml) before (open bars) and after RLN ligation (filled bars) in naive guinea pigs are shown. Vertical lines represent means ± SE; n = 4 animals per group. ***P < 0.001 compared with intact innervation.

 

Figure 6
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  		Fig. 6. CPA does not induce the release of histamine from lung mast cells. Increase in grams of tension induced by reflex tracheal smooth muscle contraction (A) and % increase in RL (B) in naive guinea pigs in response to nebulized histamine (1 mg/ml; open bars) and CPA (10 mg/ml; filled bars) before and following intravenously administered pyrilamine (1 mg/kg) are shown. Vertical lines represent means ± SE; n = 5–7 animals per group. ***P < 0.001 vs. histamine in the absence of pyrilamine.

 
Tracheal reflex: passively sensitized animals.   Baseline pulmonary function was unaffected by any of the pharmacological treatments used (data not shown). The aerosolized administration of various adenosine agonists caused a significant increase in reflex tracheal tension and airway resistance (P < 0.05, ANOVA; Fig. 7). The selective A1 agonist CPA (10 mg/ml; 10 s) induced reflex tracheal contractions in both naive and passively sensitized guinea pigs. The reflex tracheal contraction and coincident bronchoconstriction were significantly increased in passively sensitized animals when compared with naive animals (P < 0.05, Fig. 7). There was no significant difference between baseline RL in passively sensitized animals (199 ± 7.0 cmH2O·s·l–1, n = 6) compared with naive animals (192 ± 6.9 cmH2O·s·l–1, n = 14).


Figure 7
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  		Fig. 7. Reflex activation of tracheal smooth muscle in response to various adenosine receptor agonists. Increase in grams of tension induced by reflex tracheal smooth muscle contraction (A) and % increase in RL (B) in response to nebulized CGS-21680, AMP, and CPA (10 mg/ml) in naive (open bars) and passively sensitized (filled bars) guinea pigs. Vertical lines represent means ± SE; n = 3–10 animals per group. *P < 0.05 compared with naive.

 
The nonselective adenosine agonist AMP (10 mg/ml; 10 s) elicited only small tracheal reflexes in both naive and passively sensitized guinea pigs. However, AMP elicited bronchoconstriction in passively sensitized animals that was significantly greater compared with naive animals (P < 0.05, Fig. 7). The adenosine A2 agonist CGS-21680 (10 mg/ml; 10 s) also induced tracheal reflex contractions in both naive and passively sensitized guinea pigs (Fig. 7). CGS-21680 induced small changes in RL in naive and passively sensitized guinea pigs that was not significantly different (P > 0.05; Fig. 7).

A lower dose (1 mg/ml) of CPA, AMP, and CGS-21680 failed to elicit reflex bronchoconstriction (data not shown). In addition, the aerosol administration of AMP or CGS-21680 (10 mg/ml) did not significantly alter blood pressure (AMP: 40 ± 3 mmHg, n = 6; CGS-21680: 32 ± 4, n = 4) compared with untreated animals (41 ± 3 mmHg, n = 25), whereas CPA significantly reduced mean arterial blood pressure (18.7 ± 4 mmHg; P < 0.05; n = 10 vs. baseline), although it returned to baseline within the ensuing 1–2 min after administration.

Tracheal reflex: pharmacological studies in passively sensitized animals.   Atropine (1 µM), when added to the tracheal perfusate, abolished all reflex tracheal contractions to nebulized CPA (10 mg/ml), demonstrating the cholinergic nature of this response (Figs. 8 and 9).


Figure 8
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  		Fig. 8. Tracheal reflex in response to topical administration of CPA is mediated by activation of parasympathetic nerves. Increase in grams tension induced by aerosolized CPA (10 mg/ml) in the absence (A) and following (B and C) administration of atropine (1 µM) into the tracheal perfusate of an anesthetized guinea pig is shown.

 

Figure 9
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  		Fig. 9. Tracheal reflex response to CPA is increased in allergic guinea pigs. Increase in grams of tension induced by reflex tracheal smooth muscle contraction (A) and % increase in RL (B) in response to nebulized CPA (10 mg/ml) in naive (open bars) and passively sensitized (filled bars) guinea pigs. Vehicle (light gray bars) did not elicit reflex tracheal contraction or bronchoconstriction. Administration of atropine (1 µM) into the tracheal perfusate inhibited reflex contraction to nebulized CPA in passively sensitized (dark gray bar; CPA + atropine) guinea pigs. Vertical lines represent means ± SE; n = 5–9 animals per group. *P < 0.05, **P < 0.01 compared with naive.

 
Airway obstruction (% increase in RL cmH2O·s·l–1) and tracheal tension (g) in response to intravenously administered capsaicin were abolished in guinea pigs exposed chronically to capsaicin (80 mg/kg) (Fig. 10). In contrast, the cholinergic reflex reported following nebulization with CPA (10 mg/ml) was unaffected by this treatment (Fig. 10). Bradykinin (1 mg/ml) induced a small reflex tracheal contraction and bronchoconstriction; although it was reduced in animals chronically treated with capsaicin, this did not achieve statistical significance (P > 0.05; Fig. 10).


Figure 10
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  		Fig. 10. Chemical ablation of sensory C-fiber function did not impair the functional response to CPA. Increase in grams of tension induced by reflex tracheal smooth muscle contraction (A) and % increase in RL (B) in guinea pigs after 7–10 days chronic treatment with capsaicin (80 mg/kg). Changes in tension and resistance measured in response to bradykinin (1 mg/ml, light gray bars), CPA (10 mg/ml; filled bars) and capsaicin (100 µg/kg) (dark gray bar) in passively sensitized guinea pigs. Bradykinin (1 mg/ml) was administered in naive animals (open bars). Vertical lines represent means ± SE; n = 3 animals per group.

 
The intravenous administration of meclofenamic acid (3 mg/kg) did not significantly alter the increase in tracheal tone following aerosolized exposure to CPA (10 mg/ml) (tracheal tension: before 0.44 ± 0.07, n = 12 vs. 0.36 ± 0.07, n = 4). Similarly, the H1 receptor antagonist pyrilamine (1 mg/kg) also failed to significantly attenuate the reflex increase in tracheal tone induced by aerosolized CPA (10 mg/ml) in sensitized animals (0.41 ± 0.09 g; n = 5; P > 0.05 compared with control), despite causing a significant inhibition of airways obstruction (% increase in RL) to aerosolized histamine following pretreatment with pyrilamine (control: 62 ± 23, n = 3 vs. pyrilamine: 4 ± 0.2, n = 6; P < 0.05).

To further probe the mechanism of action of CPA-induced reflex tracheal contraction, the local anesthetic lidocaine (10 mg/ml; 2 min), which blocks afferent nerve endings located in the mucosal surface of the airways, was nebulized in passively sensitized guinea pigs. The reflex tracheal tension induced by nebulized CPA (10 mg/ml) was attenuated in animals exposed to aerosolized lidocaine (control: 0.58 ± 0.02 g vs. posttreatment: 0.42 ± 0.021 g; n = 3; P < 0.05).

In allergic guinea pigs, the intravenous administration of ovalbumin (100 µg/kg) caused airway obstruction (% increase in RL: 43.7 ± 9.2, n = 8) that was significantly attenuated following treatment with pyrilamine (20 ± 5; n = 5; P < 0.05). This highlighted the predominant role of histamine in mediating airway obstruction following activation of mast cells in this allergic model.

Contractility studies in passively sensitized guinea pigs.   Methacholine contracted isolated passively sensitized guinea pig trachea in a concentration dependent manner. In contrast, IB-MECA, AMP, CGS-21680, and CPA failed to contract the trachea (data not shown). However, tracheal preparations from passively sensitized animals contracted in response to 100 µM ovalbumin (64.7 ± 10.7% of methacholine maximal response, n = 5).

TRPV1 studies.   Using an in vitro cell culture system that transiently expresses TRPV1, we showed that capsaicin directly activated this receptor, which, as expected, desensitized with repeated administrations of this agonist. CPA did not activate COS7 cells expressing TRPV1 (Fig. 11).


Figure 11
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  		Fig. 11. CPA does not directly activate transient receptor potential vanilloid type 1 (TRPV1). Calcium levels measured in TRPV1 expressing cells. A: changes in intracellular calcium concentration ([Ca]i) evoked by application of capsaicin (300 nM; top) and CPA (100 µM; middle) and in combination (bottom). Bar graphs represent mean [Ca]i increases to capsaicin (B) and CPA (C). Vertical lines represent means ± SE. Number in parentheses indicates number of experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
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This study extends our laboratory's previous study (24) and clearly demonstrates that activation of adenosine receptors can induce bronchomotor responses in the airways via activation of airway sensory nerves and involve reflex tracheal smooth muscle contraction. The reflex contraction of tracheal smooth muscle to the adenosine A1 receptor agonist CPA was inhibited following treatment with atropine, ligation of RLNs, and treatment with the adenosine A1 antagonist DPCPX, but it was not inhibited following treatment with an H1 receptor antagonist. The activation of reflex contraction was still evident in animals following chronic capsaicin treatment and this highlights the involvement of non-TRPV1-positive afferent nerves in this response.

We further investigated the reflex responses in allergic animals induced by CPA to establish an allergic model that might replicate clinical observations that asthmatic but not healthy subjects bronchoconstrict to adenosine (12), a response that is inhibited by muscarinic receptor antagonism (11). The role of altered neuronal reflexes in this response was hypothesized to be the underlying basis of this clinical observation. Another important characteristic feature of an allergic model that was desirable was that the model should demonstrate sensitivity to adenosine receptor agonists (e.g., AMP, CPA) in sensitized animals that had not been subsequently challenged to the sensitizing antigen or present in healthy animals (24).

In this study, tracheal tone has been used as an indicator of parasympathetic tone throughout the airways as there is a body of evidence from functional and neuroanatomic studies in the literature to indicate that the autonomic regulation of the trachea does not significantly differ from other regions of the airways (6, 17, 31, 44). Monitoring tracheal tension is also a more sensitive method of assessing reflex responses compared with global measurements of RL. However, it is not always possible using whole lung measurements to distinguish or isolate the direct effect of stimuli on airway smooth muscle tone from that evoked by reflex induced contraction of smooth muscle. Therefore, we used a method that was optimized to detect reflex tracheal contraction while making simultaneous recordings of airway lung function (Fig. 1). The airway obstruction in response to the various agonists used was smaller in magnitude compared with our laboratory's earlier publication (24) but nonetheless were significant, and we attribute this to methodological differences in the experimental preparation.

We used CPA, a purported selective A1 agonist (Ki 2.3 nM). However, it is also clear that CPA may also stimulate A2A/A2B at higher concentrations (A2A; Ki 790 nM; A2B; Ki 18,600 nM) (26). However, the observation that a low intravenous concentration of CPA initiated reflex responses suggests that A1-receptors are likely mediating this response. In contrast, in a previous study, N6-R-phenylisopropyladenosine (R-PIA; adenosine A1 agonist) induced contraction of guinea pig trachea that was unrelated to adenosine A1 receptor activation (30). However, in our studies we show that the adenosine A1-selective receptor antagonist DPCPX (Ki 3.9 nM) (26) administered systemically inhibited CPA-mediated reflexes. Furthermore, DPCPX was present in the tracheal perfusate throughout the experiments, and so the actions of CPA are unlikely to be due to direct actions of this agonist on airway smooth muscle. Indeed, in vitro experiments using isolated tracheal tissue revealed that CPA failed to induce direct contraction of smooth muscle or to induce it indirectly via activation of mast cells. This is also consistent with the demonstration that CPA can directly activate capsaicin sensitive nodose ganglion cells using patch clamping recording techniques, and there is no reason to suggest that these receptors are not also expressed in the peripheral terminations of these neurons (10). Furthermore, there is no evidence that CPA can induce the release of acetylcholine from parasympathetic nerves, and our in vitro studies showed that tracheal smooth muscle did not respond to this agent when added exogenously, ruling out locally mediated release of acetylcholine or direct activation of airway smooth muscle. It has previously been demonstrated that adenosine ligands may directly bind to cloned and natively expressed TRPV1 (36). However, our experiments in TRPV1 transfected COS7 cells demonstrated that CPA did not directly activate TRPV1 nor interfere with the activation of this channel by capsaicin. This supports the view that CPA did not directly induce a cholinergic reflex or promote the release of sensory neuropeptides into the airways by direct activation of TRPV1.

The inhibition of the tracheal reflex response to bradykinin and CPA by atropine demonstrated that both agents induced a cholinergic reflex. Despite causing marked changes in RL, histamine produced reflex contraction of comparable magnitude to bradykinin and CPA. This shows that direct activation of airway afferent nerves by bradykinin and CPA and not contraction of smooth muscle distal to the trachea, was responsible for the activation of this reflex. This observation is consistent with previous findings that administration of histamine directly into the lung was less effective at inducing cholinergic reflex contraction of the trachea, despite marked increases in lung function (6). This hypothesis is further supported by studies showing that nodose-derived C fibers appear to be preferentially activated by bradykinin and CPA over jugular-derived C fibers (10, 41). It might be possible that a subpopulation of these C fibers innervating the intrapulmonary airways might have accounted for the atropine-resistant airways obstruction observed in response to CPA.

In passively sensitized guinea pigs, CPA-induced reflexes in the trachea were significantly increased compared with naive animals. Furthermore, CPA stimulated bronchoconstriction in sensitised guinea pigs that was absent in naive animals. This is consistent with observations in asthmatic subjects (12) and various animal models, including the allergic rabbit (13) and guinea pig (24). Although different adenosine receptors are implicated in other species (14, 18), allergic animals and asthmatic subjects respond to a greater degree to adenosine receptor agonists than nonsensitized controls or healthy subjects, respectively. The mechanism for the increased reflex response to CPA remains to be established under inflammatory conditions. However, several potential mechanisms could explain this phenomenon, and they include increased adenosine receptor expression on target cells (4); altered afferent input into the central nervous system due to an increase in neuropeptide and neurokinin receptor expression on afferent nerves and/or increased afferent activity (7, 22, 23, 32); changes in synaptic plasticity at the level of the nucleus tractus solitarius could potentially lead to an increase in the activity of second-order neurons and hence increased reflex contractile responses (8, 9); and finally, reduced function of prejunctional muscarinic M2 receptors on parasympathetic nerve terminals would lead to increased release of acetylcholine at tracheal smooth muscle (15).

Nebulized CGS-21680 (10 mg/ml), a purported selective adenosine A2A agonist, also induced reflex tracheal contraction of the trachea and increase in RL in passively sensitized guinea pigs. However, the magnitude of the reflex tracheal contractions was significantly lower than that observed with CPA. The dissociation constant values for CGS-21680 at the A2A receptor (27 nM) and for the A1 receptor (290 nM) suggest that at the concentration employed, CGS-21680 might be acting at the A1 receptor to induce tracheal reflexes. Furthermore, administration of a lower dose of CGS 21680 (1 mg/ml) failed to have effects on tracheal measurements or on lung mechanics. The possibility remains that the small reflex responses observed with CGS-21680 were via activation of the adenosine A1 receptor; alternatively, a recent study showed that CGS-21680 can also elicit the activation of nodose C fibers via adenosine A2A activation (10). However, in the present study, this activation does not appear to be sufficient to lead to airway obstruction, supporting our laboratory's previous observation (24). Moreover, there is considerable evidence showing that activation of the A2A receptor may have an anti-inflammatory action in the lung (37).

The documented evidence of adenosine A2B receptors on mast cells has focused attention on this receptor subtype in mediating the release of histamine and leukotrienes that mediate airway obstruction to adenosine in individuals with asthma (35). However, the intravenous administration of the selective H1 receptor antagonist pyrilamine did not inhibit CPA-induced tracheal reflexes or bronchospasm, suggesting that histamine release from mast cells or the activation of A2B receptors is not implicated in mediating reflex bronchospasm in passively sensitised guinea pigs. The formation of prostanoids in the airways after administration of autacoids is thought to sensitize and/or activate airway afferent nerves (25). The intravenous administration of meclofenamic acid (cyclooxygenase inhibitor) did not inhibit CPA-induced reflex tracheal contractions. Hence, prostanoid release from various cell types, including mast cells, could not account for this response. This is also consistent with an earlier study showing that bradykinin-induced cholinergic reflexes were unaffected by meclofenamic acid, whereas the bronchoconstriction was abolished by this treatment (6). Hence, prostanoid formation does not contribute significantly to cholinergic reflex responses.

The adenosine A3 receptor has been proposed to be involved in mediating AMP-induced airway obstruction in the guinea pig because in vitro experiments have shown that adenosine causes substantial contraction of sensitized guinea pig airways that was associated with the A3 receptor mechanism (30, 40). Furthermore, inhalation of AMP induced rapid migration of eosinophils and macrophages into the airways of sensitized guinea pigs that was blocked by the A3 receptor antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazo lin-5-yl] benzene acetamide (MRS-1220) (40, 43). However, IB-MECA, the adenosine A3 agonist, failed to induce reflex contraction of the trachea or increase RL when administered to passively sensitized guinea pigs, whereas CPA did induce both tracheal reflexes and bronchospasm. These data indicate that the adenosine A3 receptor is not involved in mediating reflex contraction or bronchospasm in this model.

The role for afferent nerves in mediating tracheal reflex contraction to CPA is highly persuasive. Reports from the literature show that adenosine activates pulmonary C fibers in the rat (20) and in the guinea pig (10, 29), which can be inhibited by chronic capsaicin treatment. However, in the present study, chronic capsaicin treatment did not abolish CPA-induced reflex tracheal contractions. This would suggest that CPA can also activate cholinergic reflexes independently of the activation of capsaicin-sensitive nerves, as well as activating capsaicin-sensitive nerves (24). We confirmed that chronic treatment with capsaicin had caused chemical destruction of C fibers because bronchoconstriction to capsaicin was completely abolished. Furthermore, tracheal reflex responses to bradykinin were partially inhibited in capsaicin-treated animals, which would be consistent with the ability of this agonist to activate C fibers not expressing TRPV1 (27, 28).

In summary, CPA-induced tracheal smooth muscle contraction is mediated by the reflex activation of parasympathetic nerves. This effect appeared to be mediated by direct activation of afferent nerves innervating the airway, although the contribution of mediators derived from nonneuronal cells expressing adenosine A1 receptors cannot be ruled out. However, we have established this contractile response is not secondary to airway smooth muscle contraction, prostanoid release, histamine release from mast cells, sensory neuropeptides, or direct activation of TRPV1, and it is augmented in allergic animals. Moreover, this reflex response was not inhibited by chronic capsaicin treatment. Hence, CPA can also activate TRPV1-negative afferents, consistent with the observation that adenosine activated C fibers derived from nodose and not jugular ganglion cells in the guinea pig (10). It is plausible that CPA-induced cholinergic reflexes in the guinea pig may be due to activation of TRPV1-negative neurons like the subset recently observed in TRPV1 knockout mice where bradykinin produced action potential discharge (27, 28).


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge the financial support of GlaxoSmithKline for S. M. Reynolds during her doctoral studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Spina, The Sackler Institute of Pulmonary Pharmacology, Pharmaceutical Science Research Division, School of Biomedical and Health Science, King's College London, London SE1 1UL, UK (e-mail: domenico.spina{at}kcl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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