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(Chest. 2005;128:1905-1909.)
© 2005 American College of Chest Physicians

Effects of Aerosolized Adenosine 5'-Triphosphate vs Adenosine 5'-Monophosphate on Dyspnea and Airway Caliber in Healthy Nonsmokers and Patients With Asthma*

Ozen K. Basoglu, MD; Amir Pelleg, PhD; Sarah Essilfie-Quaye, MD; Caterina Brindicci, MD; Peter J. Barnes, MA, DM, DSc, FRCP and Sergei A. Kharitonov, MD, PhD

* From the Section of Airway Diseases (Drs. Basoglu, Essilfie-Quaye, Brindicci, Barnes, and Kharitonov), National Heart and Lung Institute, Faculty of Medicine, Imperial College, Dovehouse St, London, UK; and Duska Scientific Co. (Dr. Pelleg), Bala Cynwyd, PA.

Correspondence to: Sergei A. Kharitonov, MD, PhD, Section of Airway Disease, National Heart & Lung Institute, Dovehouse St, London SW3 6LY, UK; e-mail: s.kharitonov{at}imperial.ac.uk


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Extracellular adenosine 5'-triphosphate (ATP) causes neurogenic bronchoconstriction, inflammation, and coughs, and may play a mechanistic role in obstructive airway diseases. The aims of this study were to determine the effects of inhaled ATP on airway function, and to compare these effects with those of adenosine 5'-monophosphate (AMP).

Design: Prospective, randomized, double-blind study.

Setting: Clinical research laboratory of a postgraduate teaching hospital.

Methods: The effects of inhaled equimolar doses of ATP and AMP on airway caliber, perception of dyspnea quantified by the Borg score, and other symptoms were determined in 10 nonsmokers (age 41 ± 3 years) and 10 patients with asthma (age 39 ± 3 years) [± SEM].

Results: None of the healthy nonsmokers responded to ATP or AMP. All the patients with asthma responded to ATP, and 90% responded to AMP. The geometric mean of the provocative dose causing a 20% fall in FEV1 (PD20) of ATP was 48.7 µmol/mL and that of PD20 AMP was 113.5 µmol/mL in responsive asthmatics (p < 0.05). In asthmatic patients, the percentage change in FEV1 caused by ATP was greater than that caused by AMP ({Delta}FEV1 ATP = 29% vs {Delta}FEV1 AMP = 22%, p < 0.05). Borg score increased significantly in asthmatics after ATP (from 0.1 to 3.3, p < 0.01) and after AMP (from 0.2 to 2.5, p < 0.01). This increase was also greater after ATP than AMP in asthma ({Delta}Borg ATP = 3.2 vs {Delta}Borg AMP = 2.3, p < 0.05). ATP induced cough in 16 subjects (80%), while AMP induced cough in 8 subjects (40%) [p < 0.05]; in addition, more subjects had throat irritation after inhalation of ATP than AMP (p < 0.05).

Conclusions: ATP is a more potent bronchoconstrictor and has greater effects on dyspnea and other symptoms than AMP in asthmatic patients. Therefore, ATP could potentially be used as a bronchoprovocator in the clinical setting.

Key Words: adenosine 5'-monophosphate • adenosine 5'-triphosphate • airway caliber • asthma • Borg score • dyspnea • P2 receptors


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Airway hyperresponsiveness, the characteristic physiologic abnormality of asthma, is defined as abnormal increase in both the sensitivity and magnitude of airway narrowing in response to various nonsensitizing triggers. In the clinical setting, airway responsiveness is determined by bronchial challenge using usually methacholine, histamine, or adenosine 5'-monophosphate (AMP) delivered as an aerosol. The former two compounds induce bronchoconstriction predominantly through a direct effect on airway smooth muscle, whereas AMP is believed to act indirectly by activating mast cells in the lung.1

Adenosine 5'-triphosphate (ATP) is a purine nucleotide that is found in every cell, where it plays a critical role in cellular metabolism and energetics. ATP is released from cells under physiologic and pathophysiologic conditions; extracellular ATP acts as a local physiologic regulator as well as an endogenous mediator that may play a mechanistic role in the pathophysiology of obstructive airway diseases.2 ATP exerts potent effects on dendritic cells, eosinophils, and mast cells. It enhances IgE-mediated release of histamine and other mediators from human lung mast cells.3 Extracellular ATP can also exacerbate neurogenic bronchoconstriction and inflammation by stimulating vagal sensory nerve terminals in the lungs and stimulating the release of neuropeptides.234 It has previously been shown that patients with asthma exhibit a more intense response (ie, bronchoconstriction) to inhaled ATP than normal individuals, and in both groups of subjects ATP was more potent than methacholine and histamine in reducing the baseline FEV1 by 15%.5

Adenosine is a purine nucleoside that is a product of the enzymatic degradation of ATP. Aerosolized adenosine causes bronchoconstriction in asthmatic but not healthy subjects. Since the dose responses of adenosine and AMP are identical6 and AMP is much more soluble than adenosine, AMP has been used as a bronchial challenge in the clinical setting. The effects of AMP and adenosine are largely mediated by stimulating the release of inflammatory mediators such as histamine and leukotrienes from mast cells. The quantification of airway response to AMP may be valuable in evaluating anti-inflammatory therapy and assessing disease status in relation to allergic airway inflammation.6789

Aerosolized ATP, but not AMP/adenosine, also causes bronchoconstriction in healthy subjects.5 In addition, ATP but not adenosine, activates vagal C fibers as well as the rapidly adapting receptors or A-{Delta} fibers in the airways, an action that is mediated by P2X receptors distinct from the P1 adenosine receptors.10 It seems that the actions of ATP in the lungs are independent of adenosine, the product of its enzymatic degradation. To date, the comparison of ATP and AMP challenge tests in patients with asthma has not been done. We hypothesized that the effects of inhaled ATP would be different than those of AMP. Thus, the effects of aerosolized equimolar doses of ATP and AMP on airway caliber, perception of dyspnea, and other symptoms were quantified in nonsmokers and patients with asthma.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Healthy nonsmokers (41 ± 3 years old [± SEM], n = 10) and patients with intermittent asthma (Global Initiative for Asthma guidelines) [39 ± 3 years old, n = 10] were studied (Table 1 ). Healthy nonsmokers had a normal chest examination, normal lung function without reversibility, and no history of any respiratory disorder. Asthmatic patients were clinically stable and free from respiratory tract infection or use of steroids during 4 weeks preceding the study. They were subjected to methacholine challenge test in the preceding 6 months and were all responsive to methacholine. The study was approved by the Ethics Committee of the Royal Brompton Hospital, and all subjects gave informed consent before enrollment into the study.


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  		Table 1. Characteristics of the Study Population*

 
Study Design
The study design was prospective, randomized, and double blind. Each subject attended the laboratory on three occasions. Procedures on the screening visit (first) included the recording of the medical history, lung function assessment, reversibility, and skin-prick testing to common aeroallergens. At the second and third visits, which were separated from each other by 2 to 7 days, the subjects were randomly administered either an ATP or AMP challenge. Before, immediately after, and 30 min after the challenge, spirometry was carried out, the Borg score for dyspnea was determined, and symptoms other than dyspnea were recorded.

Skin-Prick Testing
Standard skin sensitivity was measured for four common aeroallergens (house dust mite, grass pollen, cat hair, and Aspergillus fumigatus, with negative and positive controls) [Soluprick; ALK-Abello A/S; Horsholm, Denmark].

Lung Function
Spirometric and reversibility tests were performed using a dry spirometer (Vitalograph; Buckingham, UK).

Borg Score
A modified Borg scale was used, in which words describing degrees of breathlessness were anchored to numbers between 0 and 10. All subjects (responders and nonresponders to ATP/AMP) were asked to select a number whose assigned words most appropriately described their dyspnea (ie, perception of breathlessness). The change in dyspnea was also expressed as {Delta}Borg, ie, the difference in Borg score before and after the challenge.1112

Inhalation Challenge Tests
ATP and AMP were freshly dissolved in normal saline solution to produce a range of doubling concentrations from 0.227 to 929 µmol/mL (0.125 to 512 mg/mL) for ATP and from 0.138 to 1,152 µmol/mL (0.048 to 400 mg/mL) for AMP and were administrated by a breath-activated dosimeter (Mefar; Bovezzo, Italy) with an output of 10 µL per inhalation.13 While wearing a nose clip, the subjects inhaled five breaths of normal saline solution, followed by sequential doubling concentrations of either ATP or AMP. FEV1 was measured 2 min after the fifth inhalation until there was a fall in FEV1 of ≥ 20% from its value recorded after saline solution inhalation (baseline value) or until maximal concentration of either ATP or AMP was inhaled. The provocative dose causing a 20% decrease in FEV1 (PD20) was calculated by interpolation of the logarithmic dose-response curve.

Statistical Analysis
The significance of differences among groups was assessed by Student t test, and analysis of categorical variables was examined by {chi}2 test. The Pearson correlation coefficient and linear regression analysis were used to analyze the relationship between the percentage change in FEV1 and Borg score. The PD20 values for ATP and AMP were logarithmically transformed to normalize their distribution and are presented as geometric means. All other numerical variables were expressed as the mean ± SEM, and significance was defined as p < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Airway Responsiveness to AMP and ATP
None of the healthy nonsmokers responded to either ATP or AMP. All patients with asthma responded to ATP, whereas 90% of them responded to AMP, ie, showing a ≥ 20% fall in FEV1 up to the maximal concentrations administered. The geometric mean PD20 ATP was 48.7 µmol/mL (26.9 mg/mL) and PD20 AMP was 113.5 µmol/mL (39.6 mg/mL) in responsive subjects (p < 0.05) [Fig 1 ].



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  		Figure 1. Individual PD20 values of ATP (left, A) and AMP (right, B) challenge tests. Horizontal bars represent geometric mean values, and dashed lines indicate the highest concentration administrated (929 µmol/mL for ATP and 1,152 µmol/mL for AMP). The geometric mean PD20 ATP was 48.7 µmol/mL and PD20 AMP was 113.5 µmol/mL in responsive asthmatics (p < 0.05).

 
ATP-induced bronchoconstriction expressed as a percentage of the baseline FEV1 ({Delta}FEV1) was greater than that caused by AMP challenge in both groups; however, this difference was significant only in patients with asthma ({Delta}FEV1 ATP = 29% vs {Delta}FEV1 AMP = 22%, p < 0.05) [Fig 2 ].



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  		Figure 2. Percentage change in FEV1 from baseline ({Delta}FEV1) after ATP (left, A) and AMP (right, B) challenge tests. *{Delta}FEV1 was higher after ATP than AMP challenge in patients with asthma (p < 0.05).

 
Effect of ATP and AMP Challenge on Dyspnea
The perception of dyspnea assessed by the Borg score increased significantly after ATP (from 0.1 to 3.3, p < 0.01) and after AMP (from 0.2 to 2.5, p < 0.01) challenge in patients with asthma. {Delta}Borg (the difference between Borg score before and after the challenge test) after ATP was larger than after AMP challenge in patients with asthma ({Delta}Borg ATP = 3.2 vs {Delta}Borg AMP = 2.3, p < 0.05) even when the patient nonresponsive to AMP was excluded. There was a negative correlation between the concentration of PD20 and Borg score immediately after either ATP (r = – 0.85, p < 0.001) or AMP (r = – 0.88, p < 0.001) challenge when all the subjects considered together. In contrast, there was a positive correlation between {Delta}FEV1 and {Delta}Borg score both after either ATP (r = 0.82, p < 0.0001) or AMP (r = 0.77, p < 0.0001) challenge (Fig 3 ).



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  		Figure 3. The relationship between the percentage change in FEV1 from baseline ({Delta}FEV1) and {Delta}Borg score after ATP (left, A) and AMP (right, B) challenge tests in all subjects (n = 20).

 
Effect of ATP and AMP Challenge on Other Symptoms
Sixteen subjects (80%) coughed after ATP challenge, whereas AMP challenge induced cough in 8 subjects (40%) [p < 0.05]. The percentage of subjects who experienced throat irritation was also significantly higher after ATP than after AMP challenge (70% vs 35%, respectively; p < 0.05). When two groups were considered separately, it was found that the percentage of healthy subjects who had throat irritation was significantly higher after ATP vs after AMP challenge (80% vs 20%, p < 0.01). Moreover, ATP induced cough in 70% of the healthy subjects, whereas only 20% of them coughed after AMP. ATP and AMP caused cough in 90% and 60% of the asthmatics, respectively (Fig 4 ).



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  		Figure 4. Symptoms other than dyspnea after ATP (left, A) and AMP (right, B) challenge tests. The percentage of subjects who had cough and throat irritation after ATP challenge was significantly higher than AMP (*p < 0.05).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data show that ATP is a more potent bronchoconstrictor and has greater effect on dyspnea and other symptoms than AMP in asthmatic patients. Furthermore, there was a significant relationship between acute changes in airway caliber and Borg score with both ATP and AMP challenges.

All patients with asthma, who were not current smokers, responded to ATP, and 90% of them were AMP responsive. Also, ATP was 2.3-fold more potent than AMP in asthmatic patients as a bronchoconstrictor. The degree or magnitude of the subject’s response to AMP and ATP differed in asthmatic individuals. In a previous study,5 it was shown that aerosolized ATP triggered bronchoconstriction in healthy subjects and nonsmoking asthma patients; in asthmatic patients, ATP was 50-fold more potent than methacholine and 87-fold more potent than histamine in producing a 15% decrease in FEV1. Although we used similar concentrations of ATP, none of the healthy nonsmokers were responsive to ATP in our study. There is no immediate explanation for this discrepancy.

A significant increase in perception of dyspnea, as assessed by the Borg score, was observed both after ATP and AMP challenges in patients with asthma, whereas this increase was higher after ATP. Also, the Borg score correlated with the percentage fall in FEV1 after either ATP or AMP challenge. These findings are in agreement with those of Burdon et al,12 who showed that breathlessness as indicated by Borg scores increases in asthmatic subjects as the FEV1 decreases. It has been previously shown that AMP and metabisulfite, which cause bronchoconstriction by an indirect action, induce a more intense respiratory discomfort for a given fall in FEV1 than methacholine, which acts directly on airway smooth-muscle cells.14 Differential potency of ATP and AMP could be explained by the different mechanisms through which ATP and AMP cause bronchoconstriction. ATP exacerbates IgE-mediated release of histamine and other mediators from mast cells,2 basophils, and eosinophils.3 ATP also activates vagal sensory nerve terminals (C fibers) in the lungs,10 which results in reflex bronchoconstriction5 and possibly local release of neuropeptides through an axon reflex.

In this study, subjects reported more cough and throat irritation after ATP than AMP challenge; specifically, ATP and AMP triggered cough in 80% and 40% of the subjects, respectively. Since the pH of AMP (range, 3.1 to 4.3) and ATP (from 3.5 to 4.1) solutions were similar, the differential induction of cough cannot be explained by a lower pH of the ATP solution. Although cough and bronchoconstriction are believed to have separate afferent neural pathways, they often occur simultaneously and considered to be closely related. It has been demonstrated that ATP given as a rapid bolus in either the right atrium or the pulmonary arteries of dogs stimulates both vagal pulmonary capsaicin-sensitive C-fibers10 as well as rapidly adapting receptors (A-{Delta} fibers) [unpublished data; A. Pelleg; June 2003]. The latter are known to play a major mechanistic role in cough, and the former play a facilitating role. However, adenosine, a breakdown product of ATP, did not mimic the action of ATP on the canine pulmonary vagal C-fibers.10

Our results indicate that the inhalation of ATP has more potent effects than AMP on perception of dyspnea, induction of cough, and bronchoconstriction. Based on the present and previously published data, it is tempting to speculate that extracellular ATP and P2 receptors play a mechanistic role in obstructive airway diseases by activating airway sensory nerves that may be sensitized in patients with obstructive airway diseases. Better understanding of the role of ATP in airway diseases, may lead to novel therapies based on selective blockade of specific purinoceptor subtypes in the lung.


   Footnotes
 
Abbreviations: AMP = adenosine 5'-monophosphate; ATP = adenosine 5'-triphosphate; PD20 = provocative dose causing a 20% fall in FEV1

This study was supported by Duska Scientific Co., Bala Cynwyd, PA.

Received for publication December 22, 2004. Accepted for publication April 5, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cain, H (2001) Bronchoprovocation testing. Clin Chest Med 22,651-659[Medline]
  2. Pelleg, A, Schulman, ES Adenosine 5'-triphosphate axis in obstructive airway diseases. Am J Ther 2002;9,454-464[CrossRef][Medline]
  3. Schulman, ES, Glaum, MC, Post, T, et al ATP modulates anti-IgE-induced release of histamine from human lung mast cells. Am J Respir Cell Mol Biol 1999;20,530-537[Abstract/Free Full Text]
  4. Katchanov, G, Xu, J, Schulman, ES, et al ATP causes neurogenic bronchoconstriction in the dog. Drug Dev Res 1998;45,342-349[CrossRef]
  5. Pellegrino, R, Wilson, O, Jenouri, G, et al Lung mechanics during induced bronchoconstriction. J Appl Physiol 1996;81,964-975[Abstract/Free Full Text]
  6. Polosa, R, Rorke, S, Holgate, ST Evolving concepts on the value of adenosine hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Thorax 2002;57,649-654[Abstract/Free Full Text]
  7. Van Schoor, J, Joos, GF, Pauwels, RA Indirect bronchial hyperresponsiveness in asthma: mechanisms, pharmacology and implications for clinical research. Eur Respir J 2000;16,514-533[Abstract]
  8. van den Berge, M, Polosa, R, Kerstjens, HA, et al The role of endogenous and exogenous AMP in asthma and chronic obstructive pulmonary disease. J Allergy Clin Immunol 2004;114,737-746[Medline]
  9. Lee, DK, Gray, RD, Lipworth, BJ Adenosine monophosphate bronchial provocation and the actions of asthma therapy. Clin Exp Allergy 2003;33,287-294[CrossRef][Medline]
  10. Pelleg, A, Hurt, CM Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals. J Physiol 1996;490,265-275[ISI][Medline]
  11. Rutgers, SR, ten Hacken, NH, Koeter, GH, et al Borg scores before and after challenge with adenosine 5'-monophosphate and methacholine in subjects with COPD and asthma. Eur Respir J 2000;16,486-490[Abstract]
  12. Burdon, JG, Juniper, EF, Killian, KJ, et al The perception of breathlessness in asthma. Am Rev Respir Dis 1982;126,825-828[ISI][Medline]
  13. Chai, H, Farr, RS, Froehlich, LA, et al Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol 1975;56,323-327[CrossRef][ISI][Medline]
  14. Marks, GB, Yates, DH, Sist, M, et al Respiratory sensation during bronchial challenge testing with methacholine, sodium metabisulphite, and adenosine monophosphate. Thorax 1996;51,793-798[Abstract]



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