Volume 116, Issue 2 , Pages 312-317, August 2005
Endobronchial adenosine monophosphate challenge causes tachykinin release in the human airway
Article Outline
Background
Adenosine 5 monophosphate (AMP) has been shown to cause bronchoconstriction and a sensation of chest tightness when inhaled by asthmatic subjects. This response is attenuated after repeated inhalation of bradykinin, suggesting that AMP may act in part by the release of neuropeptides.
Objective
This study examined neuropeptide release in the human airway after endobronchial AMP challenge.
Methods
Endobronchial AMP challenge was performed in 20 subjects and tachykinin levels were measured after endobronchial AMP challenge and after placebo endobronchial challenge with saline.
Results
All subjects coughed immediately after adenosine challenge. There was a significant increase in neurokinin A and substance P levels (P<.01, P<.01 respectively) when post-saline and post-AMP levels were compared. There was, however, no significant change in calcitonin gene related peptide levels (P=.37).
Conclusion
This study demonstrates that endobronchial AMP challenge causes tachykinin release in the human airway in vivo.
Key words: Tachykinin, neuropeptide, adenosine, endobronchial challenge
Abbreviations used: AMP, Adenosine 5′ monophosphate, CGRP, Calcitonin gene related peptide, NEP, Neutral endopeptidase, NKA, Neurokinin A, NKB, Neurokinin B, NPK, Neuropeptide K, PC20 AMP, Provocative concentration of AMP causing a 20% fall in FEV1, SP, Substance P
Adenosine is a naturally occurring purine nucleoside that functions as a constituent of nucleic acid, as an intracellular and autocoid mediator.1 Elevated levels have been found in bronchoalveolar lavage fluid of asthmatic subjects as compared to normal subjects, suggesting that adenosine may be a mediator in asthma.2 Inhalation of adenosine monophosphate (AMP), which is rapidly dephosphorylated to adenosine in vivo,3 causes bronchoconstriction in atopic asthmatic and non-asthmatic subjects but not in non-atopic, non-asthmatic subjects.4 The related nucleoside guanosine has no effect, suggesting that this is a specific receptor mediated effect.5
Atopic asthmatic and non-asthmatic subjects cough and bronchoconstrict in response to stimuli such as inhaled AMP and sulphur dioxide; however, asthmatics tend to respond to lower concentrations.6 Cough and chest tightness are both common symptoms in asthma and are related to stimulation of sensory nerves.6
Evidence exists that while AMP acts mainly via primed mast cells, the agent also stimulates vagal nerves. Pre-treatment with ipratropium (an anti-cholinergic agent) has a bronchoprotective effect on the response to AMP, suggesting activation of cholinergic nerves in the response to AMP.7, 8 Inhalation of AMP and bradykinin cause a greater sensation of chest tightness than does inhalation of methacholine, for the same degree of bronchoconstriction, suggesting that the former acts on sensory pathways.9 Repeated inhalation of bradykinin attenuates the response to inhaled AMP suggesting that both of these agents act in part via liberation of neuropeptides from sensory nerves.10
Hong et al11 have shown that pulmonary C fibers (the nerve fibers containing neuropeptides) in the rat are activated after right atrial injection of adenosine, implicating these nerves in the response to AMP in this model. The purpose of this study was to examine neuropeptide release in vivo in the human airway after endobronchial AMP challenge.
Methods
Subjects
Ethical approval was granted by the Research Ethics Committee of the Queen's University of Belfast. All subjects gave written informed consent. All subjects were non-smokers and had not received any anti-histamines or inhaled or oral steroids in the preceding six months. Asthmatics were recruited if they (1) had a prior clinical diagnosis of asthma and a history of intermittent shortness of breath or wheeze, (2) were atopic (reacted to at least one allergen on skin prick testing), and (3) had FEV1 greater than 60% predicted. All other subjects had no symptoms suggestive of asthma; atopic non-asthmatic subjects had at least one positive skin prick test as aforementioned.
All subjects attended on two occasions. At the screening visit, informed consent was obtained and clinical assessment, skin prick testing, and AMP inhalation challenge were performed. At the subsequent visit (at least 72 hours after screening visit), bronchoscopy and endobronchial AMP challenge were performed.
Skin prick testing
Skin prick testing was performed using a standardized puncture technique,12 using allergen preparations of house dust mite, cat, and dog protein and grass pollen (Dome-Hollister-Stier, Epernon Cedex, France). A positive reaction was taken as a wheal size of 3 mm or more.
Inhalational challenging
Spirometry was performed according to American Thoracic Society Guidelines13 using a Vitalograph spirometer (Buckingham, UK). AMP (Sigma-Aldrich Ltd., Poole, UK) was freshly prepared in 0.9% saline in doubling concentrations ranging from 0.391 mg/mL to 400 mg/mL. The AMP provocation test was performed using the two-minute tidal breathing method of Cockcroft et al14 using a Medix Turbonebuliser (Leicestershire, UK) with an output of 0.13 mL/min. PC20 AMP was calculated by linear interpolation.
Bronchoscopy
AMP was freshly made up on the morning of the bronchoscopy in 0.9% saline from a stock solution of 400 mg/mL. At bronchoscopy, subjects were given intravenous Midazolam (up to 14 mg) to achieve mild sedation and the hypopharynx was anaesthetised using 4% lignocaine spray. Vocal cord and tracheal anaesthesia was achieved using 4 mL of 4% lignocaine introduced trans-cricoidally. Oxygen was routinely applied at 2 L/min via nasal cannulae. Heart rate, ECG, and oxygen saturations were monitored throughout the procedure. The bronchoscope (240 IT Olympus Optical Co. Ltd. Tokyo, Japan) was introduced orally and 2-mL aliquots of 2% lignocaine were used as necessary to anesthetize the airways below the carina to suppress coughing.
The site of the subsequent endobronchial challenge was randomized prior to bronchoscopy. Subjects were randomly assigned a number, which determined the site of the active challenge to either the right middle or upper lobes, and randomization was constrained to achieve balance. The placebo challenge was automatically assigned to the opposite site from the active challenge.
The bronchoscope was initially wedged in a segmental orifice of the site randomized for the placebo challenge and a baseline bronchial wash with 20 mL of saline was performed and aspirated back after minimum dwell time. A placebo challenge of 5 mL of saline was administered to the same segment and the segment closely observed for any visible reaction. After 3 minutes, a second bronchial wash using 20 mL of saline was performed and aspirated back after minimal dwell time.
The active (AMP) challenge was then performed in the other site. Again a baseline bronchial wash was performed using 20 mL saline and immediately aspirated back under gentle suction. Then the active challenge with 5 mL AMP was performed. The initial AMP concentration administered was one tenth that which caused a 20% fall in FEV1 on the prior inhalational challenge or if the subject had been unresponsive to adenosine one tenth of the maximum concentration during the inhalation challenge (400 mg/mL). Up to two subsequent AMP doses were given at quadrupling concentrations, the maximum administered endobronchial dose being 400 mg/mL. There was a time lapse of 3 minutes after each concentration given to observe for any visual reaction using the analogue outlined in Table I. The endobronchial challenge was terminated either when there was a visible reaction to AMP, when the maximum concentration of AMP had been administered or when it was necessary to terminate the challenge for reasons of patient comfort. Three minutes after the final concentration of adenosine had been administered, a further bronchial wash of 20 mL of saline was performed and aspirated after a minimum dwell time. Subjects remained under observation for a period of at least two hours after the procedure.
Table I. Visual analogue reaction for grading of response after endobronchial challenge
| Analogue score | Reaction observed |
|---|---|
| 0 | No reaction |
| 1 | Subject coughs after instillation of AMP (no coughing after saline challenge) |
| 2 | Subject coughs/immediate bronchial pallor then hyperemia/increased mucus secretion after instillation of AMP |
| 3 | Bronchoconstriction observed after instillation of AMP |
Processing of samples
A total cell count was measured using a modified Neubauer hemocytometer and was expressed as the number of cells ×105/mL of BAL. Cell viability was assessed by Trypan blue exclusion staining. Viable cells are expressed as a percentage of total cell numbers. Samples were centrifuged at 200 × g for 10 minutes at 4°C to separate any debris and added to a protease inhibitor cocktail (see Appendix) and stored at −70°C for subsequent analysis.
Neuropeptide measurement
NKA was measured using radioimmunoassay, utilizing a N-terminal specific anti-serum that was raised in guinea pigs to synthetic human NKA (Amersham Bioscience UK Ltd product number IM168, Buckinghamshire, UK). It cross-reacts fully with NKB and NPK but less than 0.1% with SP. The detection limit for the assay is 2 ng/L.
CGRP immunoreactivity was measured using a commercial CGRP human radioimmunoassay (RIA) kit (catalogue number RIK009, Peninsula Laboratories, San Carlos, Calif). This antibody is a rabbit anti-human CGRP peptide (II) antibody. The label was 125I-Tyr0-CGRP (catalog number Y6011). The limit of detection for this assay is 2 ng/L and the antibody cross-reacts 100% with human CGRP (II), human CGRP, and rat CGRP. It cross-reacts <0.001% with rat calcitonin C-terminal adjacent peptide and less than 0.02% with insulin, glucagon, somatostatin, SP, vasoactive intestinal peptide, and gastrin releasing peptide.
Substance P (SP) was measured using a commercially available ELISA (catolog number DE1400, R&D Systems, Minneapolis, Minn). It shows no significant cross-reactivity with NKA, neurokinin B (NKB), and neuropeptide K (NPK). The limit of detection of this assay is 8 pg/mL.
For radioimmunoassays, lavage fluid was extracted using a previously validated technique.16 In brief, cleared plasma and traysolol were added to equal volume of lavage fluid followed by precipitation of large molecular weight proteins in 60% alcohol and the sample centrifuged (30 min, × 1500 g). Thiomersal was added to the supernatant. This was then decanted, the extract was evaporated to dryness and the sample assayed. For SP ELISA, samples were Sep-pakked (C18 Sep-pak; Waters, Milford, Mass) and eluted using a 60:40 solution of acetontirile and 1% trifluoro-acetic acid, dried down and reconstituted in buffer prior to assay. Using these extraction and assay methods, peptide recovery was >90%. We have previously characterised NKA, SP, and CGRP immunoreactivity in bronchoalveolar lavage fluid using HPLC, confirming it to be target peptide.17
Measurement of osmolality of AMP solutions
AMP was freshly made up in 0.9% normal saline in concentrations ranging from 0.39 mg/mL to 400 mg/mL and osmolality measured (Advanced Micro-osmometer 3300; Advanced Instruments Inc, Pomona, Calif).
Statistical analysis
All statistical analysis was performed using SPSS version 11.0 (Statistical Package for Social Sciences, Chicago, Ill). All data were tested for normality of distribution using Shapiro-Wilk W tests. Where the data were skewed values are quoted as median and interquartile range (IQR), unless otherwise stated. For non-parametric data, comparisons were made between paired samples using the Wilcoxon analysis and for unpaired samples using the Mann-Whitney U test. Comparison between more than two groups were performed using Kruskall-Wallis analysis. Post hoc multiple comparisons were then performed to demonstrate the underlying statistical differences (Stats Direct, Cambridge, UK). Non–parametric correlations were calculated using Spearman's rank correlations. Subjects who did not achieve at least a 20% drop in FEV1 after inhaling 400 mg/mL of AMP were given an assigned PC20 value of 640 mg/mL for statistical analysis. P values <.05 were regarded as statistically significant.
Results
A total of 24 subjects were recruited. Three subjects (1 normal, 1 atopic, and 1 asthmatic) did not complete the endobronchial challenge. Twenty-one subjects completed the endobronchial challenge protocol (1 sample was inadequate for processing). The demographic details for the remaining 20 subjects (7 normals, 6 atopic non-asthmatics, and 7 atopic asthmatics) are shown in Table II. There was no difference between the groups in terms of age or baseline spirometry.
Table II. Demographic details of subjects
| Normals (N) | Atopics (AT) | Asthmatics (AS) | |
|---|---|---|---|
| Number | 7 | 6 | 7 |
| Age (y), Median (IQR) | 21 (21-24) | 21 (21-24) | 26 (21-30) |
Sex (n =male) | 4 | 3 | 2 |
| FEV1% predicted, Mean (SD) | 114.0 (8.4) | 99.8 (14.2) | 90.0 (14.8) |
| FVC% predicted, Mean (SD) | 97.7 (11.0) | 99.2 (15.4) | 95.6 (16.4) |
| PC20 AMP (mg/mL), Median (IQR) | 640.0 | 413.9 (144.8-640.0) | 3.6 (2.3-7.9) |
| Maximum dose of endobronchial AMP (mg/mL), Median (range) | 160.0 (40.0-400.0) | 40.0 (10.0-160.0) | 1.3 (0.15-10.0) |
| Number of aliquots of endobronchial AMP, Median (range) | 2 (1.0-3.0) | 1 (1.0-2.0) | 1 (1.0-3.0) |
Measurements of osmolality of saline and AMP solutions showed the following: 0.9% saline, 281 mOsm; 0.39 mg/mL, 282 mOsm; 3.125 mg/mL, 297 mOsm; 12.5 mg/mL, 326 mOsm; 40 mg/mL, 403 mOsm; 100 mg/mL, 567 mOsm; 400 mg/mL, 1292 mOsm.
Endobronchial responses to AMP challenge are shown in Figure 1. Instillation of endobronchial AMP led to immediate coughing in all subjects after administration of adenosine, which was not seen after instillation of the saline placebo challenge. However, there was no evidence of generalised lung involvement (eg, wheeze, hypoxia) in response to the endobronchial AMP challenge in any subject.
Fig 1.
The frequency of endobronchial response to AMP challenge in normals, atopics, and asthmatics.
As immediate coughing was observed in all groups, for the purposes of analysis all subjects were considered together. There was a significant increase in median (IQR) NKA and substance P levels [10.0 (5.0–15.0) vs. 20.0 (10.6–25.0) pg/mL, P < .01 and 227. 8 (176.9–278.6) vs. 318.1 (190.6–422.9) pg/mL, P < .01] respectively when post-saline and post-AMP levels were compared (Fig 2). There was no significant change in CGRP levels (P=.37, Fig 2). There was no significant difference between groups in NKA, CGRP, or substance P levels at baseline or post-challenge.
Fig 2.
The change in post-saline and post-AMP levels of (A) NKA, (B) Substance P, and (C) CGRP.
There was no correlation between the change (post-AMP–post-saline) in NKA and substance P after AMP challenging (r=0.27, P=.25) or between the change in CGRP and substance P (r=0.25, P=.34). There was no significant correlation between the change in NKA or substance P and the PC20 AMP for the group as a whole (r=0.09, P=.71, r=0.26, P=.31 respectively). There was no difference between the changes in NKA observed between those who bronchoconstricted after endobronchial challenge (visual reaction 3) and those who did not (P=.30).
Discussion
This study provides the first evidence of in vivo tachykinin release after a chemical airway challenge in humans. During the study it was observed that many of the subjects who were sedated and who did not cough during the placebo saline challenge or baseline lavage at the active challenge site, started to cough immediately after the instillation of endobronchial AMP. In three cases this coughing induced by AMP was believed to be distressing enough to necessitate termination of the procedure.
The immediacy of the cough after AMP challenge suggests that the cough is mediated by afferent sensory nerves. It has previously been shown that AMP acts on vagal nerves.7, 8, 9 The co-existent increase in NKA and substance P suggest the mechanism may involve stimulation of sensory C fibres with antidromic release of tachykinins.
Stimulation of sensory C fibers and release of neuropeptides have previously been implicated in cough. It is known that inhalation of capsaicin acting through the VR-1 receptor causes cough.18, 19 Capsaicin also liberates neuropeptides from sensory nerves and after repeated inhalation the cough diminishes, possibly due to depletion of neuropeptides and suggesting propagation of cough by neuropeptides in this model. There is evidence, in animal models, that AMP can act directly on pulmonary C fibers11; however, the exact receptor involved seems to vary between species. In rats it was found that adenosine potentiated the response of pulmonary C fibers to chemical stimuli and that this response was attenuated by pre-treatment with an A1 receptor antagonist20; while in guinea pig lung A2 agonists decreased the release of SP from pulmonary C fibers.21 Thus, it is possible in this study that AMP was acting directly on nerve endings to cause cough via a central mechanism rather than directly by neuropeptide release, although this potential mechanism has not been well studied in the human airway.
The coughing after endobronchial AMP challenge and subsequent release of tachykinins was observed across all groups of subjects. While it has been suggested that the nerves of asthmatics may be more sensitive to stimuli than those of non-asthmatics, the asthmatics in this study were mild both in terms of airway hyperresponsiveness and treatment requirements and may not reflect differences that may occur with more significant asthma.
There was no correlation between the changes in NKA, CGRP, or substance P. This is perhaps not surprising in the case of NKA, as this neuropeptide may be located independently of the other two peptides.22 However, it is surprising that there was no correlation between the changes seen in substance P and CGRP because they are often co-localized in the same neurones. There are a number of possible explanations for this discrepancy. While neuropeptides are co-localized the exact amounts present may vary depending on the location of the tissue.23 There may also be differential paths of degradation. Substance P and NKA are primarily degraded in the airways by the enzyme neutral endopeptidase (NEP). However, the exact degradation pathway of CGRP has not yet been elucidated. It has been shown that CGRP is subject to degradation by tryptase while the tachykinins NKA and substance P are not.24 Most of the subjects in this study had tryptase release after endobronchial AMP challenging,15 which may explain why there was no difference in CGRP levels after AMP challenge and also why there is no relationship between CGRP and SP levels. In addition, it may be that differential release accounts for the patterns of neuropeptide release observed. The relative amount of neuropeptide released may be dependent on the frequency with which the nerve is stimulated as well as the quantity of each peptide in the nerve ending.25 However, the precise mechanisms remain to be established.
Another possibility is that while AMP mediates the cough through afferent sensory nerves, the tachykinin release is from a non-neuronal source. Sensory nerves containing neuropeptides account for around 1% of all the nerve fibers found in human airways.22 However, much higher levels of neuropeptides have been found in induced sputum 26 and in BAL17 than would be expected from this source alone. It has been shown that tachykinins may be produced by eosinophils, macrophages, lymphocytes, neutrophils, and epithelial cells,27, 28, 29, 30 and it may be that these non-neuronal sources are responsible for the neuropeptide release induced by AMP in this study. Thus, the neuropeptide release and the cough may be two concomitant but separate events in the airway after AMP challenge.
Nasal challenge with hypertonic saline induces neuropeptide release31 and mannitol challenge in asthmatics causes cough that is independent of bronchoconstriction.32 Osmolality of the adenosine concentrations demonstrated that the challenges used in normal subjects were relatively hyperosmolar compared to normal saline/plasma; however, this was not the case for the concentrations used in asthmatic subjects (Table II). Thus, while we cannot completely exclude an osmotic effect in normal subjects, given this mechanism is not applicable in asthmatic subjects, we believe it is unlikely that two entirely separate mechanisms are producing cough and causing tachykinin release.
There was no relationship between PC20 AMP and the change in NKA, SP, or CGRP levels. Bronchoconstriction after inhalation of AMP is mediated by multiple mechanisms including the release of mast cell mediators, which were present in many of these subjects.15 Thus a simple correlation between changes in airway physiology and individual endobronchial tachykinin or other mediator release would seem unlikely.
This study has provided evidence that endobronchial AMP challenge causes immediate cough and significant NKA and substance P release in non-atopic non-asthmatic, atopic non-asthmatic, and atopic asthmatic subjects, which occurs in the absence of significant CGRP release. This is the first demonstration of in vivo tachykinin release, after chemical stimulation, in the human airway. It therefore seems likely that AMP can act through a number of mechanisms, in addition to mast cell mediator release, to generate responses in the human airway.
Appendix
Protease inhibitor cocktail contained the following reagents at recommended concentrations:
References
- Release of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med. 1995;151:624–629
- . Adenosine in bronchoalveolar lavage fluid in asthma. Am Rev Respir Dis. 1993;148:91–97
- . Airway effects of purine nucleosides and nucleotides and release with bronchial provocation in asthma. J Appl Physiol. 1986;61:1667–1676
- . Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br J Clin Pharmacol. 1983;15:161–165
- . The action of purines on human airways. Arch Int Pharmacodyn Ther. 1986;280(2 Suppl):240–252
- . Airway sensory nerves: a burning issue in asthma?. Thorax. 1996;51:335–337
- . Protective effects of inhaled ipratropium bromide on bronchoconstriction induced by adenosine and methacholine in asthma. Eur Respir J. 1992;5:560–565
- . The effect of inhaled ipratropium bromide alone and in combination with oral terfenadine on bronchoconstriction provoked by adenosine 5′- monophosphate and histamine in asthma. J Allergy Clin Immunol. 1991;87:939–947
- Respiratory sensation during bronchial challenge testing with methacholine, sodium metabisulphite, and adenosine monophosphate. Thorax. 1996;51:793–798
- . Repeated inhalation of bradykinin attenuates adenosine 5′-monophosphate (AMP) induced bronchoconstriction in asthmatic airways. Eur Respir J. 1992;5:700–706
- . Activation of pulmonary C fibres by adenosine in anaesthetized rats: role of adenosine A1 receptors. J Physiol. 1998;508:109–118
- Skin tests used in type I allergy testing Position paper. Sub-Committee on Skin Tests of the European Academy of Allergology and Clinical Immunology. Allergy. 1989;44(Suppl 10):1–59
- Standardization of spirometry–1987 update. Statement of the American Thoracic Society. Am Rev Respir Dis. 1987;136:1285–1298
- . Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy. 1977;7:235–243
- . Mast cell mediator release in non-asthmatic subjects after endobronchial adenosine challenge. J Allergy Clin Immunol. 2004;114:34–39
- . Radioimmunological determination of pancreatic and gut glucagon in plasma. Diabetologia. 1971;7:10–19
- . Neurokinin A is the predominant tachykinin in human bronchoalveolar lavage fluid in normal and asthmatic subjects. Thorax. 1998;53:357–362
- . Sensory neuron -specific actions of capsiacin: mechanisms and applications. Trends Pharmacol Sci. 1990;11:330–333
- . The capsaicin receptor: a heat activated ion channel in the pain pathway. Nature. 1997;389:816–824
- . Hypersensitivity of pulmonary C fibres induced by adenosine in anaesthetized rats. J Appl Physiol. 2003;95:1315–1324
- Effects of adensoine A2 receptor agonists on the excitation of capsaicin-sensitive afferent sensory nerves in airway tissues. Eur J Pharmacol. 1993;240:121–126
- . Role of tachykinins in asthma. Allergy. 2000;55:321–337
- . Neurokinin A and substance P vary independently in different regions of rat sensory neurons. Neuropeptides. 1995;28:237–241
- . Mast cell tryptases and airway remodeling. Am J Respir Crit Care Med. 2001;164:S52–S58
- . Autonomic regulation. i-NANC/e-NANC. Am J Respir Crit Care Med. 1998;158:S171–S175
- Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med. 1995;151:613–617
- . The effects of tachykinins on inflammatory and immune cells. Regul Pept. 1997;70:75–90
- . Neural mechanisms in asthma. Clin Exp Allergy. 2000;30(Suppl 1):60–65
- . Airway inflammation and tachykinins: prospects for the development of tachykinin receptor antagonists. Eur J Pharmacol. 2001;429:239–250
- . Pre-protachykinin-A mRNA is increased in the airway epithelium of smokers with chronic bronchitis. Respirology. 2001;6:187–197
- . Hypertonic saline nasal provocation stimulates nocioceptive nerves, substance P release and glandular mucus exocytosis in normal humans. Am J Respir Crit Care Med. 1999;160:655–662
- . Coughing during mannitol challenge is associated with asthma. Chest. 2004;125:1985–1992
Funding: Northern Ireland Chest Heart and Stroke Association.
PII: S0091-6749(05)00612-3
doi:10.1016/j.jaci.2005.03.034
© 2005 American Academy of Allergy, Asthma and Immunology. Published by Elsevier Inc. All rights reserved.
Volume 116, Issue 2 , Pages 312-317, August 2005
