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. 2005 Jan 31;144(7):1011–1016. doi: 10.1038/sj.bjp.0706133

Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge

Kristin N Kornerup 1, Clive P Page 1, James D Moffatt 1,*
PMCID: PMC1576083  PMID: 15685200

Abstract

  1. The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated.

  2. Mucosally applied adenosine caused increases in short-circuit current (ISC) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (ΔISC 69.5±6.7 μA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in ISC, which were not investigated further.

  3. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM–10 μM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxoamido adenosine; CGS 21680; 0.1–100 μM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide; IB-MECA; 30 nM–100 μM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine.

  4. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM–1 μM) caused a rightward shift of the adenosine concentration–effect curve only at 1 μM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration–effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors.

  5. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline ISC was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency.

  6. These results suggest that the A2B receptor mediates the ISC response to adenosine in the mouse trachea. This receptor does not appear to be regulated in a standard asthma model.

Keywords: Adenosine, ion transport, airway, allergy

Introduction

Extracellular purine nucleotides and nucleosides signal via two families of receptors, adenosine (P1) receptors and P2 receptors. The two families can be distinguished pharmacologically by the purines that activate them (Ralevic & Burnstock, 1998; Bucheimer & Linden, 2004). Thus, adenosine acts primarily at adenosine (A1, A2A, A2B, A3) receptors, whereas other purines such as ATP, ADP and UDP potently activate P2 receptors. P2 receptors are further divided into G-protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels (Ralevic & Burnstock, 1998; Bucheimer & Linden, 2004). Purinoceptors have been identified at most epithelial surfaces and modulate epithelial ion transport in the gastrointestinal system, kidney and lung (Bucheimer & Linden, 2004). ATP has been demonstrated to increase chloride conductance by the airway epithelium via P2Y2 receptors (Cressman et al., 1999; Kellerman et al., 2002; Bucheimer & Linden, 2004). Release of ATP by airway epithelial cells has also been demonstrated, suggesting that ATP may act in an autocrine or paracrine manner (Homolya et al., 2000).

Adenosine is the breakdown product of ATP via endogenous ecto-ATPases and is also present at cell surfaces in cultured airway epithelial cells (Lazarowski et al., 2004). However, the pharmacology of the effect of adenosine on ion transport by the epithelium of the lower airways has rarely been investigated. Adenosine has been demonstrated to increase chloride secretion in human primary cultures of nasal epithelium as well as in transformed and other epithelium-like cell lines (Lazarowski et al., 1992; Cobb et al., 2002). Although the effect of adenosine on cultured human lower airway epithelial cells has been demonstrated previously (Lazarowski et al., 2004), a thorough pharmacological analysis has not been performed. Furthermore, data from freshly isolated preparations are completely lacking. Thus, the primary aim of the present study was to determine which receptor(s) mediates ion transport changes in the mouse isolated trachea, a species now widely used in respiratory research.

Adenosine is also an interesting mediator as it is a provocative challenge that selectively causes bronchoconstriction in asthmatics only (Barnes et al., 1998). There has been interest in adenosine and regulation of adenosine receptors in asthmatics and animal models of the disease (El-Hashim et al., 1999; Fozard et al., 2002), including those in mice (Fan et al., 2003). Therefore, a second aim of the present study was to establish if the effect of adenosine on ion transport was affected by allergic sensitisation and challenge.

Methods

Animals and experimental procedure

All experiments were carried out under The Animals (Scientific Procedures) Act, 1986. Female BALB/c mice (Harlan, Bichester, Oxon., U.K.), 6–8 weeks old were killed with an overdose of urethane (20 g kg−1 i.p.) and the entire trachea was removed and opened longitudinally along the dorsal surface to form a flat sheet. This sheet was then mounted between two perspex Ussing chambers (with a surface area of 0.06 cm2), which contained a modified Krebs solution (composition (mM): NaCl 118; KCl 5.4; MgSO4 0.57; glucose 11; KH2PO4 1.2; NaHCO3 25; CaCl2 2.5), which was maintained at 37°C and continuously bubbled with 95% O2/5% CO2. Current and voltage electrodes (containing 4% agar in 2 M KCl; World Precision Instruments; Stevenage, Herts., U.K.) attached to the chambers were connected to an amplifier (DVC 1000, World Precision Instruments), which was then used to voltage clamp the tissue at 0 mV. Changes in short circuit current (ISC) were used as a measure of the effect of adenosine and other drugs on the preparation.

After a 30 min equilibration period, the maximum secretory response of each preparation was determined by adding ATP (100 μM; lumenal side), which from preliminary experiments was found to be the most efficacious secretogogue in this preparation. The peak response to maximum concentrations of ATP (100 μM) in preliminary experiments was 69.5±6.7 μA cm−2, greater than substance P (1 μM; 45.7±4.3), bradykinin (1 μM; 13.2±2.2) and adenosine (100 μM; 38.8±5.2; all luminal, n=7 for each). All responses to adenosine and other agonists are expressed as a percentage of this maximal ATP response. After washout of ATP, the preparations were allowed to equilibrate for a further 30 min before the addition of agonists. Preliminary experiments established that adenosine caused monophasic increases in ISC, which reached a plateau that was steady for >30 s, allowing cumulative concentration–effect curves to be established. The symmetry of these curves did not indicate that receptor desensitisation was occurring during agonist addition. However, in some preparations a second curve, repeated up to an hour after the initial curve was depressed or slightly right-shifted. Therefore, for the purposes of this study only one concentration–effect curve was performed on each tissue. Where the effects of antagonists were examined, these were added after the washout of ATP.

Allergic sensitisation and challenge

On day 1, mice were sensitised with an i.p. injection of 10 μg chicken egg ovalbumin (Sigma, Poole, Dorset, U.K.) per mouse in 0.1 M Al2(OH)3 adjuvant in saline and boosted in the same way 7 days later. Control mice were sham sensitised and boosted with alum/saline only. On days 14–17, the animals were exposed to a 30 min aerosol challenge of ovalbumin (1 mg ml−1). This protocol consistently produces lung eosinophilia and airway hyper-responsiveness in this laboratory (Riffo-Vasquez et al., 2000; Pitchford et al., 2004). Tissues were isolated for study on day 18 as described above.

Statistics

Concentration–effect curves were fitted by nonlinear regression using the computer programme Graphpad Prism (Version 2.01; Graphpad Software, San Diego, CA, U.S.A.) to determine pEC50 values. Statistical differences between these values were assessed using one-way analysis of variance with Newman–Keuls post-test. Student's t-tests were used to compare differences between control and immunised animals. A P-value of less than 0.05 was considered significant.

Drugs

Adenosine hemisulphate, ATP, N6-cyclopentyladenosine (CPA) and 5′-N-ethyl-carboxamidoadenosine (NECA) were purchased from Sigma. 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxoamido adenosine hydrochloride (CGS 21680), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), 1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were purchased from Tocris Cookson (Avonmouth, Bristol, U.K.).

Adenosine and NECA were prepared as 100 mM solutions in distilled water. All other drugs were prepared as 100 mM stocks in DMSO and subsequently diluted in water, with the exception of IB-MECA, which was diluted in 50% DMSO to 1 mM and subsequently in distilled water. The volumes of DMSO added to the bath did not alone cause significant changes in ISC.

Results

Effect of adenosine on ISC

In four preparations, the effect of adenosine on ISC when applied to the adventitial side of the preparation was examined. Three preparations responded with small (range 13–18% of ATP) increases in ISC with a pEC50 of 4.68±0.22 (Figure 1a). These small responses were not investigated further.

Figure 1.

Figure 1

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(a) Comparison of the effect of cumulatively added adenosine on ISC when applied to the lumenal or adventitial surface of the isolated mouse trachea (n=4–5). Responses have been normalised to the response of the tissue to ATP, the most efficacious secretogogue in this preparation. (b) Comparison of several mucosally applied adenosine analogues in this assay (n=4–6). The concentration–effect curve for adenosine is reproduced from panel (a) in panel (b).

Adenosine caused increases in ISC reaching a maximum of approximately 50% of ATP in all preparations studied when added to the lumenal surface of the preparation (Figure 1b). With the exception of CGS 21680, all adenosine analogues tested caused ISC changes with similar efficacy, although potencies varied (Figure 1b; Table 1). The nonselective adenosine receptor agonist NECA was the most potent, the A1 selective agonist CGS 21680 the least potent, while IB-MECA and CPA had similar potencies to adenosine. Thus, the rank order of potencies for adenosine and adenosine analogues in this preparation is NECA>adenosine=CPA=IB-MECA>CGS 21680.

Table 1.

pEC50 and maximum response values for adenosine and adenosine analogues in the Ussing chamber assay

Compound pEC50 % ATP max n
Adenosine 5.11±0.2 53±7 6
NECA 6.24±0.3 51±7 4a
CPA 5.73±0.1 47±3 4
IB-MECA 5.42±0.2 56±6 4
CGS 21680 4.63±0.2 30±5 4a
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a

Significantly different to the potency of adenosine, P<0.05 (one-way analysis of variance with Newman–Kuels post-test)

Effects of adenosine receptor antagonists

The A1-selective antagonist DPCPX (10 nM–1 μM) caused a rightward shift of the adenosine concentration–effect curve only at the highest concentration tested (Figure 2a). The A2A-preferring, mixed A2A/A2B antagonist ZM 241385 (0.3–10 μM) shifted the adenosine concentration–effect curve to the right with an apparent pA2 of 7.2 (Figure 2b and c). Neither DPCPX nor ZM 241385 altered baseline ISC at concentrations that affected the adenosine concentration–effect relationship.

Figure 2.

Figure 2

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Effect of adenosine receptor antagonists on cumulative concentration–effect curves for adenosine in the mouse trachea. (a) The A1-selective antagonist DPCPX had no effect on the concentration–effect relationship at concentrations less than 1 μM (n=3–6). (b) Dose-dependent rightward shift of the adenosine concentration-effect curve by the A2A/A2B antagonist ZM 241385 (n=4–5). (c) Schild analysis of the effect of ZM 241385 on the adenosine concentration–effect curve, demonstrating a slope close to unity. The pA2 derived from this analysis was 7.2.

Effect of allergen challenge on responses to adenosine

Preparations from allergic animals had a significantly lower baseline ISC (Figure 3a) and produced smaller responses to ATP (Figure 3b), suggesting that secretion was impaired. There was no difference in the potency of adenosine between sensitised (pEC50=5.10±0.26) and sham-sensitised (pEC50=5.26±0.15) mice, and when normalised to the response to ATP, there was no difference in efficacy (Figure 3c).

Figure 3.

Figure 3

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Effect of allergic sensitisation and 3 days of aerosol challenge on the ISC response of the murine trachea to adenosine. (a) Unstimulated ISC was significantly lower in the allergic compared with sham-sensitised controls (n=5 each). (b) The increase in ISC following ATP addition (100 μM) was also significantly attenuated in the allergic group (n=5 each). (c) Normalised to the response to ATP, there was no difference in the potency or efficacy of the response to adenosine between the two groups (n=5 each). *Indicates a significant difference (P<0.05) between the sham and allergic groups (unpaired t-test).

Discussion and conclusions

The present study confirms that, like ATP, adenosine is a secretogogue in the normal, freshly isolated airway epithelium. This response appears to be mediated by adenosine receptors of the A2B subtype, based on the potencies both of agonists and antagonists for these receptors. However, there does not appear to be any significant regulation of this receptor in an acute model of allergic airway disease.

Adenosine can act at four distinct receptor subtypes: A1, A2A, A2B and A3. Agonists with known increased potency relative to adenosine (Klotz, 2000) at A1 (CPA), A2A (CGS 21680) and A3 (IB-MECA) were either equipotent or less potent than adenosine, suggesting that none of these receptors mediate the action of adenosine in this preparation. Indeed the rank order of agonists (NECA>Adenosine=CPA=IB-MECA>CGS 21680) is similar to that reported for A2B receptors in other tissues such as human coronary arteries (Kemp & Cocks, 1999) and rat mesenteric artery (Prentice et al., 1997), as well as in receptor expression systems (Patel et al., 2003). The results of studies using relatively selective receptor antagonists provide further evidence that A2B receptors mediate the response to adenosine. Thus, the A1 selective antagonist DPCPX, which antagonises A1 receptors in the nanomolar range (e.g. pA2 9.3–9.7 in the rat duodenum; Nicholls & Hourani, 1997; see also Klotz, 2000), caused rightward shifts of the adenosine concentration–effect curve only at micromolar concentrations, in agreement with studies of A2B receptors in other systems (Fozard et al., 2003). Similarly, the A2A-selective antagonist ZM 241385 caused rightward shift of the adenosine concentration–effect curve only at micomolar concentrations with a pA2 of 7.2, consistent with the known potency of this compound at A2B receptors in several species (Poucher et al., 1995; Ongini et al., 1999; Fozard et al., 2003). Therefore, both agonist and antagonist studies support a role for A2B in the mouse tracheal epithelium. However, the effect of ZM 241385 at high concentrations, where increased concentrations of this antagonist appeared to fail to further right-shift the adenosine concentration–effect curve, may indicate the presence of a low affinity, non-A2B-binding site. Nevertheless, the predominant adenosine receptor in this preparation appears to be a A2B, and as cultured human bronchial epithelial cells have been suggested to express A2B receptors coupled to ion secretion (Lazarowski et al., 2004), mice may be a useful model for in vivo experimentation in this area.

Inhaled adenosine selectively causes bronchoconstriction only in asthmatics and adenosine receptors have been implicated in asthma and are a potential target for future therapies (Barnes et al., 1998; Fozard, 2003). Airways from asthmatics have increased numbers of mucus-secreting cells, and in fatal asthma mucus is frequently found to be plugging the airways (Jeffery, 2003; Cohn et al., 2004). Since ion transport by the epithelium maintains the sol layer upon which mucus is moved by ciliated epithelial cells, any dysfunction in ion transport would impact on mucus clearance (Boucher, 2003). In this and a previous study (Cloutier et al., 2004), aerosol challenge of sensitised mice with allergen resulted in a decreased baseline ISC and blunted responses to secretogogues. Whether this is a feature of the human disease does not appear to have been investigated, despite the important role of ion transport in the functioning of the mucocilliary escalator that must cope with an increased mucous burden in asthma. In a recent study, nasal epithelium was used as a surrogate for bronchial epithelial secretory responsiveness, and no difference between control and mild asthmatics was observed (Chung et al., 2003). Further studies with cells isolated from bronchial biopsies might reveal more detail about possible dysfunction of ion transport in asthmatics. In culture, two important cytokines that are involved in the allergic response, IL-4 (Galietta et al., 2002) and IL-13 (Danahay et al., 2002), cause a hypersecretory phenotype in human airway epithelial cells and both of these cytokines have been repeatedly shown to be present and important in conventional acute murine allergic models (Elias et al., 2003) where we observed a hyposecretory effect of allergen challenge. Furthermore, in the intestine during an immune response to nematode infection in mice, in addition to hypersecretion due to reduced glucose transport, responses to secretory agonists are impaired (Madden et al., 2004), suggesting that alterations in ion transport may be a common feature of mucosal surfaces following antigen challenge. It will be important to determine whether the opposite effects of these cytokines on ion transport between murine and human cells is due to a species difference or the experimental conditions (in vitro vs in vivo).

Of concern to the present studies is how to interpret the effect of the allergic response on the response to adenosine when baseline and stimulated ISC appears to be downregulated in the allergic animals. As raw ISC measurements, the response to adenosine is significantly blunted, yet if the data are normalised to the response to ATP – as we have presented them – then there is no difference between the maxima of the curves. Irrespective of whether the data are normalised or not, the potency of adenosine remained unchanged. Thus, while whole lung A2B receptor mRNA expression increases in murine asthma models, and A2B receptors may contribute to bronchomotor responses to adenosine in allergic mice (Fan et al., 2003), no change in A2B receptor function is observed in the airway epithelium. These findings suggest that the regulation of A2B receptor expression is cell-specific during the acute inflammatory response to allergen.

In unstimulated cultured human airway epithelial cells, endogenous adenosine has recently been shown to activate A2B receptors tonically (Lazarowski et al., 2004). In the present study, however, A2B antagonists had no effect on baseline ISC. This difference is probably due to the huge difference in the volumes bathing the preparations in each study. Thus, in the present study each side of the preparation was bathed by 5 ml of physiological saline and the surface area of the preparation was 0.06 cm2. By contrast, very small volumes of media – mimicking the airway surface liquid layer – were used in the cell culture model. It would be of interest to determine whether A2B antagonists have any effect on baseline transepithelial ion fluxes in vivo.

In conclusion, mucosally applied adenosine causes an increase in ISC in the mouse isolated trachea that is mediated by adenosine A2B receptors. However, in a well-characterised allergic murine model, there was no change in the potency of adenosine in producing this effect, although the baseline and stimulated ISC was impaired in allergic animals.

Abbreviations

CPA

N6-cyclopentyladenosine

DPCPX

8-cyclopentyl-1,3-dipropylxanthine

B-MECA

1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide

NECA

5′-N-ethyl-carboxamidoadenosine

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