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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2002 Feb;135(3):685–696. doi: 10.1038/sj.bjp.0704516

Evidence for an atypical receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitized Brown Norway rats

J P Hannon 1, B Tigani 1, C Wolber 1, I Williams 1, L Mazzoni 1, C Howes 2, J R Fozard 1,*
PMCID: PMC1573180  PMID: 11834616

Abstract

  1. The bronchoconstrictor response to adenosine is markedly and selectively increased following ovalbumin (OA) challenge in actively sensitized, Brown Norway rats. We present a pharmacological analysis of the receptor mediating this response.

  2. Like adenosine, the broad-spectrum adenosine receptor agonist, NECA, induced dose-related bronchoconstriction in actively sensitized, OA-challenged animals. In contrast, CPA, CGS 21680 and 2-Cl-IB-MECA, agonists selective for A1 A2A and A3 receptors, respectively, induced no, or minimal, bronchoconstriction.

  3. Neither the selective A1 receptor antagonist, DPCPX, nor the selective A2A receptor antagonist, ZM 241385, blocked the bronchoconstrictor response to adenosine.

  4. MRS 1754, which has similar affinity for rat A2B and A1 receptors, failed to block the bronchoconstrictor response to adenosine despite blockade of the A1 receptor-mediated bradycardia induced by NECA.

  5. 8-SPT and CGS 15943, antagonists at A1, A2A, and A2B but not A3 receptors, inhibited the bronchoconstrictor response to adenosine. However, the degree of blockade (approximately 3 fold) did not reflect the plasma concentrations, which were 139 and 21 times greater than the KB value at the rat A2B receptor, respectively.

  6. Adenosine and NECA, but not CPA, CGS 21680 or 2-Cl-IB-MECA, induced contraction of parenchymal strip preparations from actively sensitized OA-challenged animals. Responses to adenosine could not be antagonized by 8-SPT or MRS 1754 at concentrations >50 times their affinities at the rat A2B receptor.

  7. The receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in actively sensitized BN rats cannot be categorized as one of the four recognized adenosine receptor subtypes.

Keywords: Adenosine receptors, airway hyperresponsiveness, allergen challenge, Brown Norway rat, lung parenchymal strip

Introduction

We have recently described a marked and selective augmentation of the bronchoconstrictor response to adenosine following allergen challenge in actively sensitized, Brown Norway (BN) rats (Fozard & Hannon, 2000; Hannon et al., 2001). The response occurs against a background of mild pulmonary inflammation (Palser et al., 2000), exhibits tachyphylaxis (Fozard & Hannon, 2000) and is primarily, if not exclusively, a consequence of mast cell activation (Hannon et al., 2001). In all these respects it bears similarity to the bronchoconstrictor response to inhaled adenosine seen in asthmatic patients (Phillips & Holgate, 1995; Jacobson & Bai, 1997; Marquardt, 1997; Polosa & Holgate, 1997; Feoktistov et al., 1998; Forsythe & Ennis, 1999; Meade et al., 2001).

The receptor mediating the bronchoconstrictor response to adenosine in asthmatics has not been established, although evidence is available implicating the A2B receptor (Feoktistov et al., 1998; Fozard & Hannon, 1999). The aim of the present work was to characterize the receptor(s) mediating the bronchoconstrictor response to adenosine, augmented following allergen challenge, in actively sensitized BN rats. To this end, the effect of allergen challenge on the sensitivity of the airways to a range of subtype selective adenosine receptor agonists was defined both in vivo and in vitro. In addition, the effects of a series of adenosine receptor antagonists, some of them subtype selective, on the augmented bronchoconstrictor response to adenosine were investigated.

A part of these results has been presented to the British Pharmacological Society (Hannon et al., 1999a, 1999b; Fozard et al., 2001).

Methods

Animals

Male BN rats weighing 200 – 300 g were supplied by Biological Research Laboratories (Füllinsdorf, Switzerland). They were kept at an ambient temperature of 22°C under a 12 h normal phase light-dark cycle and fed on NAFAG® pellets supplied by Nahr und Futtermittel AG, Gossau, Switzerland. Drinking water was freely available. All experiments were carried out with the approval of the Veterinary Authority of the City of Basel (Kantonales Veterinaeramt, Basel-Stadt).

Sensitization and challenge with allergen

The procedure is based on that described by Tarayre et al. (1992). Ovalbumin (OA; 20 μg ml−1) was mixed (30 min on ice) in a blender (Polytron, Kinematica Ltd.) with aluminium hydroxide (20 mg ml−1) and injected (0.5 ml per animal s.c.) coincidentally with Acullulare pertussis adsorbat vaccine (0.2 ml per animal i.p.; diluted 1 : 4 with saline 0.9% w v−1). Injection of OA, together with adjuvant, was repeated 14 and 21 days later. Sensitized animals were used in experiments between days 28 and 35. For challenge with OA, animals were briefly anaesthetized (4% isofluran) in an anaesthetic chamber. OA or vehicle (saline, 0.2 ml per animal) was administered intratracheally (i.t.) and the animals allowed to recover.

Measurement of lung function

Animals were anaesthetized with sodium pentothal (70 mg kg−1 i.p.) and a tracheotomy performed. Heparinized polyethylene catheters were inserted into the left carotid artery for recording blood pressure and into the left jugular vein for drug administration. To suppress spontaneous respiration animals were given an intramuscular injection of vecuronium bromide (12 mg kg−1). No experiment lasted longer than 90 min, during which time surgical anaesthesia was maintained without the need for supplementary anaesthesia. Body temperature was maintained at 37°C with a heated pad controlled by a rectal thermistor.

Animals were ventilated (7 ml kg−1, 1 Hz) via the tracheal cannula with a mixture of air and oxygen (50 : 50) in order to maintain the pO2, pCO2 and pH of the blood within the physiological range. Ventilation was monitored at the trachea by a pneumotachograph (Fleisch 0000, Zabona, Switzerland) in line with the respiratory pump and connected to a differential pressure transducer (MP 4514871, Validyne, U.S.A.). Coincident pressure changes within the thorax were measured via an intrathoracic cannula, using a differential pressure transducer (MP 4524, Validyne, U.S.A). From measurements of airflow and transpulmonary pressure, airway resistance (RL, cm H2O l−1 s−1) was calculated after each respiratory cycle by use of a digital electronic pulmonary monitoring system (PMS, Mumed, London, U.K.). Mean arterial blood pressure (MABP) and heart rate (HR; by derivation) was recorded from the carotid artery by means of a pressure transducer (P23Dd, Gould, U.S.A).

In vivo studies: experimental protocols

Agonist analyses

Responses to the different adenosine receptor agonists were determined 3 h following challenge of actively sensitized animals with either vehicle (saline, 0.2 ml i.t.) or OA (0.3 mg kg−1 i.t.). This dose of OA and the pretreatment time of 3 h give a marked and consistent increase in the bronchoconstrictor response to adenosine (Hannon et al., 2001). Agonists were given by bolus i.v. injection, and to avoid tachyphylaxis only one response was generated per animal. The agonists used were 5′-N-ethylcarboxamido adenosine (NECA), a broad spectrum adenosine receptor ligand (Bruns et al., 1986), N6-cyclopentyladenosine (CPA), a selective A1 receptor ligand (Londos et al., 1980), 2-[p-(2-carboxylethyl) phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), a selective A2A receptor ligand (Jarvis et al., 1989; Wan et al., 1990), and 2-chloro-N6-(3-iodobenzyl) 5′-N-methylcarboxamidoadenosine (2-Cl-IB-MECA), an agonist with high affinity and selectivity for the A3 receptor (Kim et al., 1994). The doses used were derived from the literature where they have been shown to give cardiovascular effects in rats consistent with the subtype selectivity of the agonist in question (Fozard & Carruthers, 1993; van schaick et al., 1996).

Antagonist analyses

Three hours following challenge with OA (0.3 mg kg−1 i.t.), animals were given a bolus i.v. injection of one of the following antagonists; the broad spectrum adenosine A1, A2A, A2B receptor antagonists, 8-(p-sulphophenyl) theophylline (8-SPT; Jacobson et al., 1999) and 9-chloro-2,2-(furanyl)[1,2,4]triazolo [1,5-c]quinazolin-5-amine (CGS 15943; Kim et al., 1998), the A1 receptor selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; Bruns et al., 1987; Jacobson et al., 1999), the A2A receptor selective antagonist, 4-(2-[7-amino-2-(2-furyl) [1,2,4] triazolo [2,3-a][1,3,5] triazin-5-yl amino]ethyl) phenol (ZM 241385; Palmer et al., 1995; Poucher et al., 1995; Kim et al., 1998) the A2B receptor selective antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS 1754; Kim et al., 2000), or their respective vehicles. Five min later animals were given adenosine (1 mg kg−1) by bolus i.v. injection and 15 min later a dose-response curve to 5-HT (3 – 30 μg kg−1 i.v.) was performed; the interval between doses was 2 min. After a further 5 min a dose-response curve to an adenosine receptor agonist corresponding to the selectivity and/or dose of the antagonist utilized was established. Thus, CPA was used in conjunction with DPCPX, CGS 21680 with ZM 241385 and NECA with 8-SPT, CGS 15943 and MRS 1754.

Adenosine receptor antagonist concentrations in plasma

Animals were anaesthetized with sodium pentobarbitone (60 mg kg−1 i.p.) and one femoral vein and carotid artery cannulated. Compounds were given i.v. and blood samples were taken after 2, 5, 10, 20 and 30 min. Plasma was prepared by centrifugation and the drug concentrations measured by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).

Measurement of histamine and 5-HT in plasma

Actively sensitized animals were challenged with OA (0.3 mg kg−1, i.t.) and 2.5 h later anaesthetized with sodium pentothal (70 mg kg−1 i.p.). Polyethylene catheters were placed in both the left carotid artery (for blood collection) and right jugular vein (for drug administration). After set-up, the animals were left for a stabilization period of at least 20 min. Three hours after OA challenge animals were given a bolus injection of either 8-SPT (40 mg kg−1) or vehicle (saline, 1 ml kg−1). One min later a blood sample was taken from the carotid artery to give the baseline value. Five min later, animals were given adenosine (1 mg kg−1) by bolus i.v. injection and 1 min later a second blood sample was taken. Blood samples (approximately 1 ml) were taken into 1.5 ml ethylenediaminetetraacetate-coated plastic collection tubes and chilled on ice. Samples were immediately centrifuged (1700–g, 30 min, 4°C; Omnifuge 2.0, Heraeus Sepratech, CH) and the overlying plasma aspirated and stored at −30°C prior to assay. The concentrations of histamine and 5-HT in the plasma were measured by colorimetric assay using commercial kits (Immunotech) as previously described (Hannon et al., 2001).

In vitro studies

Lung parenchymal strip

The method is described in detail in Hannon et al. (2001). In brief, Brown Norway rats, sensitized to OA as described above, were challenged with OA (0.3 mg kg−1) or vehicle (saline) 3 h prior to death by exposure to carbon dioxide. The lungs were perfused in situ with modified Krebs' solution of the following composition (mM): NaCl 118; KCl 4.8; MgSO4, 1.2; CaCl2 2.5; KH2PO4, 1.2; NaHCO3, 25; glucose 11, prior to slices being prepared and set up for recording isotonic tension. After a stabilization period of 1 h, during which time the tissues were repeatedly washed, a supramaximal concentration of bethanechol (0.1 mM) was added. After repeated washing during 1 – 1.5 h, a single concentration of adenosine or an adenosine receptor agonist was added to the bath, followed 1 h later, after repeated washing, by establishment of a dose-response curve to 5-HT (100 nM – 0.1 mM) and, a further 1 h later, a curve to bethanechol (100 nM – 0.1 mM). Tension changes were expressed relative to the maximal response to bethanechol. In the antagonist studies, compounds were included in the Krebs' solution from 15 min prior to establishment of a response to adenosine (1 mM) for the duration of the experiment.

Longitudinal muscle from colon

The distal colon was rapidly removed from BN rats killed by exposure to carbon dioxide and placed in modified Krebs' solution of the composition described above. The longitudinal muscle and the muscularis mucosa were separated as described by Bailey & Hourani (1992). Segments of longitudinal muscle (10 mm long, 3 mm wide) were set up for recording isotonic tension in 10 ml organ baths containing modified Krebs' solution at 37°C bubbled with 95% O2/5% CO2. Resting tension was maintained at 0.5 g. After a stabilization period of 1 h, during which time the tissues were repeatedly washed, a supramaximal concentration of bethanechol (0.1 mM) was added to the bath. After repeated washing during 1 – 1.5 h, bethanechol (0.1 mM) was added to the bath followed again by washout. The tissue was then exposed a third time to bethanechol (0.1 mM) followed 20 min later by establishment of a relaxant concentration-response curve to NECA (10 nM – 0.1 mM) in the presence of the bethanechol. After repeated washing, a second concentration-response curve to NECA in the presence of bethanechol was established. Antagonists or vehicle (dimethylsulphoxide (DMSO), final bath concentration 0.2%) were included in the Krebs' solution 30 min prior to establishment of the second concentration-response to NECA. Relaxant effects were expressed relative to the maximal response to bethanechol. The dissociation constant of the antagonist-receptor complex, KB, was calculated from the equation: Inline graphic; where Inline graphic is the ratio of concentrations of agonist giving an equal response in the presence and in the absence of a given concentration of antagonist, B (Furchgott, 1972).

Materials

Aluminium hydroxide was from Merck, Germany. Acullulare pertussis adsorbat vaccine was from the Vaccinal and Serotherapic Institute of Bern, Switzerland. Pentothal (thiopentalum natricum) and Forene (isofluran, 100%) were obtained from Abbott, Switzerland. Norcuron (vecuronium bromide) was from Organon Teknika, Holland. Ovalbumin was obtained from Fluka, Switzerland. Bethanechol (carbamyl-β-methyl-choline chloride), 5-hydroxytryptamine creatinine sulphate and adenosine hemisulphate were obtained from Sigma, Switzerland. 9-Chloro-2,2-(furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS 15943), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 8-p-(sulphophenyl) theophylline (8-SPT), 5′-N-ethylcarboxamidoadenosine (NECA), 2-[p-(2-carboxyethyl)phenylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), N6-cyclopentyladenosine (CPA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyl-carboxamide (2-Cl-IB-MECA) were obtained from Research Biochemicals International, U.S.A. 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) was a gift from Astra Zeneca Pharmaceuticals, U.K. N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS 1754) was synthesized at Novartis Horsham Research Centre U.K. 8-SPT was made up in saline, (NaCl 0.9% w v−1) solution. The adenosine receptor agonists, DPCPX and ZM 243185 were dissolved at 1 mg ml−1 in 50% DMSO in distilled water and diluted immediately before use in saline. CGS 19543 and MRS 1754 (at the 1 mg kg−1 dose) were dissolved in a solution of DMSO, Sandimmun® placebo solution (Novartis Pharma AG, Switzerland) and distilled water (1 : 4 : 5). For the 3 mg kg−1 dose of MRS1754, the vehicle was polyethyleneglycol 200 containing 2% DMSO.

Data analysis

All data are presented as means±s.e.mean. Statistical analysis was performed on raw data by means of Student's t-test for paired data or analysis of variance with post hoc pairwise multiple comparison procedures, using SigmaStat for Windows, version 2.03. A P value <0.05 was considered significant.

Results

Effect of subtype selective adenosine receptor agonists in actively sensitized BN rats challenged with OA

In actively sensitized BN rats challenged with vehicle (saline, 0.2 ml i.t.) 3 h previously, the broad-spectrum adenosine receptor agonist, NECA (3 – 100 μg kg−1 i.v.) induced dose-dependent bronchoconstrictor responses. In animals challenged with OA (0.3 mg kg−1 i.t.), responses to NECA were markedly and significantly augmented (Figure 1). NECA also induced falls in both MABP and HR, reflecting its broad range of adenosine receptor activities. Unlike the effects on the airways, the cardiovascular effects were poorly dose-related, a consequence of even the lowest dose of NECA inducing close to a maximum change in both MABP and HR (Table 1).

Figure 1.

Figure 1

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The bronchoconstrictor effects of adenosine, NECA, CPA, CGS 21680 and 2-Cl-IB-MECA in actively sensitized, Brown Norway rats 3 h post intratracheal instillation of vehicle (saline, 0.2 ml) or ovalbumin (OA, 0.3 mg kg−1). The agonists were given i.v. and to avoid tachyphylaxis, only one response was generated per animal. Results are expressed as means±s.e. means (n=4 – 5). **P<0.01, ***P<0.001 that the value is significantly different from the equivalent value in the vehicle-challenged group. For corresponding cardiovascular data, see Table 1.

Table 1.

Effects of adenosine agonists on mean arterial blood pressure (MABP) and heart rate (HR) in actively sensitized Brown Norway rats, challenged intratracheally 3 h previously with either vehicle (saline, 0.2 ml) or ovalbumin (OA, 0.3 mg kg−1)

graphic file with name 135-0704516t1.jpg

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The selective A1 receptor agonist, CPA (10 and 30 μg kg−1 i.v.), induced profound bradycardia, and hypotension which was maximal and similar in both vehicle- and OA-challenged animals (Table 1). Thus, the adenosine A1 receptor is activated at these doses. CPA produced only weak bronchoconstrictor responses in animals challenged with vehicle. Similarly, in the OA-challenged animals bronchoconstrictor responses were small, although at the higher dose (30 μg kg−1) a significant increase relative to the vehicle-treated animals was observed (Figure 1).

The selective A2A receptor agonist, CGS 21680 (30 μg kg−1 i.v.), induced a prominent hypotensive response indicating that the dose was adequate to activate the A2A receptors of the systemic circulation to induce vasodilatation (Table 1). Despite this, CGS 21680 did not induce bronchoconstrictor effects following either vehicle- or OA-challenge (Figure 1).

The selective A3 receptor agonist, 2-Cl-IB-MECA (40 – 1000 μg kg−1 i.v.), induced similar pronounced, dose-related hypotensive responses in animals challenged with vehicle or OA (Table 1), consistent with activation of A3 receptors. In animals challenged with vehicle, 2-Cl-IB-MECA induced little or no effect on airway resistance. In contrast, in animals challenged with OA, 2-Cl-IB-MECA induced small bronchoconstrictor responses at the two highest doses. Although significant relative to the effects in vehicle-challenged animals, the bronchoconstrictor responses were markedly less than those seen with either adenosine or NECA (Figure 1).

Effect of adenosine receptor antagonists on the augmented response to adenosine induced by allergen challenge in actively sensitized BN rats

Broad spectrum adenosine receptor antagonists

8-SPT (20 and 40 mg kg−1 i.v.), induced dose-dependent inhibition of the augmented bronchoconstrictor response to adenosine in actively sensitized, OA-challenged animals, by 55 and 82%, respectively. By reference to the dose response relationship in Figure 1, such inhibition represents an approximate 3 and 8 fold blockade of the response to adenosine, respectively. Bronchoconstrictor responses to 5-HT were similar in both 8-SPT and vehicle-treated animals. Testifying to the non-selective antagonist properties of 8-SPT, both the bradycardia and hypotension induced by adenosine were blocked dose-dependently by 8-SPT as were the cardiovascular effects of subsequent administration of NECA (Figure 2). CGS 15943 (1 mg kg−1 i.v.) induced a significant, 62%, inhibition (corresponding to an approximate 3 fold blockade) of the bronchoconstrictor response to adenosine, with no blockade of the bronchoconstrictor response to 5-HT. Both the hypotension and bradycardia in response to adenosine and NECA were markedly and significantly reduced by CGS 15943 (Figure 3).

Figure 2.

Figure 2

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Effect of 8-SPT (20 – 40 mg kg−1, given i.v. 5 min prior to adenosine), on responses to i.v. administration of adenosine, 5-HT and NECA, with respect to airway resistance, mean arterial blood pressure (MABP) and heart rate (HR) in actively sensitized Brown Norway rats 3 h post intratracheal instillation of ovalbumin (0.3 mg kg−1). Means±s.e.means are presented (n=4 – 8). ***P<0.001 that the value is significantly different from the equivalent value in the vehicle-treated group.

Figure 3.

Figure 3

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Effect of CGS 15943 (1 mg kg−1, given i.v. 5 min prior to adenosine) on responses to adenosine, 5-HT and NECA), with respect to airway resistance, mean arterial blood pressure (MABP) and heart rate (HR) in actively sensitized Brown Norway rats 3 h post intratracheal instillation of ovalbumin (0.3 mg kg−1). Means±s.e.means are presented (n=5). **P<0.01, ***P<0.001 that the value is significantly different from the equivalent value in the vehicle-treated group.

Selective adenosine receptor antagonists

The selective A1 receptor antagonist, DPCPX (100 μg kg−1 i.v.), had no effect on the augmented bronchoconstrictor response to adenosine, nor did it affect the bronchoconstrictor responses to 5-HT (Figure 4). Clear evidence that the dose of DPCPX was sufficient to block the A1 receptor comes from the observation that the bradycardia induced both by adenosine and by the selective A1 receptor agonist, CPA, was markedly and significantly suppressed in these animals (Figure 4). The selective A2A receptor antagonist, ZM 241385 (30 μg kg−1 i.v.), had no effect on the bronchoconstrictor response to adenosine augmented following OA-challenge. Responses to the two highest doses of 5-HT were significantly reduced following ZM 241385 (Figure 5). That the dose of ZM 241385 was sufficient to establish adenosine A2A receptor blockade in these animals was demonstrated by the marked inhibition of the hypotensive response induced by the selective A2A receptor agonist, CGS 21680 (Figure 5).

Figure 4.

Figure 4

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Effect of DPCPX (100 μg kg−1, given i.v. 5 min prior to adenosine) on responses to adenosine, 5-HT and CPA), with respect to airway resistance, mean arterial blood pressure (MABP) and heart rate (HR) in actively sensitized Brown Norway rats 3 h post intratracheal instillation of ovalbumin (0.3 mg kg−1). Means±s.e. means are presented (n=5). **P<0.01, ***P<0.001 that the value is significantly different from the equivalent value in the vehicle-treated group.

Figure 5.

Figure 5

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Effect of ZM 241385 (30 μg kg−1, given i.v. 5 min prior to adenosine) on responses to adenosine, 5-HT and CGS 21680, with respect to airway resistance, mean arterial blood pressure (MABP) and heart rate (HR) in actively sensitized Brown Norway rats 3 h post intratracheal instillation of ovalbumin (0.3 mg kg−1). Means±s.e. means are presented (n=5). *P<0.05, **P<0.01 that the value is significantly different from the equivalent value in the vehicle-treated group.

The selective A2B receptor antagonist, MRS 1754, given at a dose of 1 mg kg−1 i.v. had no significant effects on the airway or cardiovascular responses to adenosine, 5-HT or NECA (Figure 6A). Similarly, at the 3 mg kg−1 dose, MRS 1754 had no significant effects on the bronchoconstrictor effects of adenosine although the responses to the lower doses of 5-HT were suppressed (Figure 6B). The vehicle used to deliver the 3 mg kg−1 dose caused some loss of sensitivity to the cardiovascular effects of NECA (see Figure 6). Despite this, both the fall in blood pressure, and in particular the bradycardia, induced by adenosine and NECA were significantly inhibited (Figure 6).

Figure 6.

Figure 6

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Effect of MRS 1754 (1 and 3 mg kg−1, given i.v. 5 min prior to adenosine, represented in panels A, B, respectively) on responses to adenosine, 5-HT and NECA, with respect to airway resistance, mean arterial blood pressure (MABP) and heart rate (HR) in actively sensitized Brown Norway rats 3 h post intratracheal instillation of ovalbumin (0.3 mg kg−1). Means±s.e. means are presented (n=4 – 5). *P<0.05, **P<0.01, ***P<0.001 that the value is significantly different from the equivalent value in the vehicle-treated group.

Histamine and 5-HT concentrations following adenosine administration: effects of 8-SPT

In agreement with previous results (Tigani et al., 2000; Hannon et al., 2001), adenosine (1 mg kg−1) administered i.v. to actively sensitized animals challenged 3 h previously with OA, increased the plasma concentrations of histamine and 5-HT in the 5 min period following injection. The increases in histamine and 5-HT were significantly inhibited by pretreatment with 8-SPT (40 mg kg−1 i.v., 5 min prior to adenosine) by 51 and 69%, respectively (Figure 7).

Figure 7.

Figure 7

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Changes in histamine (upper panel) or 5-HT (lower panel) plasma concentrations induced by adenosine (ADO, 1 mg kg−1 i.v.) in actively sensitized Brown Norway rats challenged 3 h previously with ovalbumin: Effects of pretreatment with 8-SPT (40 mg kg−1 i.v.), or vehicle (saline, 1 ml kg−1 i.v.), given 5  min prior to adenosine. Each column represents the mean±s.e.mean of four animals. #P<0.05, ##P<0.01, ###P<0.001 indicates significant difference between pre- and post-adenosine values in the same animals. *P<0.05 indicates significant difference between post adenosine values in vehicle- and 8-SPT-treated animals.

Plasma concentrations of antagonists: relationship to affinity for rat A1, A2B and A3 receptors

The data are shown in Table 2. The plasma concentrations of 8-SPT, CGS 15943 and MRS 1754 measured 5 min after i.v. administration of 20, 1 and 1 mg kg−1 respectively (corresponding to the time when adenosine was administered in the functional studies) were high, exceeding the KB values at the A2B receptor present in rat colon longitudinal muscle strip by 139, 21 and 184 fold, respectively. Similarly, the plasma concentrations of 8-SPT, CGS 15943 and MRS 1754 at the time of starting the NECA dose response curve exceeded the KB values at the A1 receptor (literature values) by 7, 16 and 27 fold, respectively.

Table 2.

The plasma concentrations of adenosine receptor antagonists, following i.v. administration to anaesthetized Brown Norway rats. Relationship to their affinities for rat adenosine receptor subtypes.

graphic file with name 135-0704516t2.jpg

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In vitro studies on lung parenchymal strips

Lung parenchymal strips taken from sensitized animals challenged with OA manifest concentration-dependent constrictor responses to adenosine which reflect the in vivo findings, in that they are augmented relative to the responses obtained on strips from vehicle-challenged animals and that the response is mast cell mediated (Hannon et al., 2001). NECA also induced contractions of strips taken from animals challenged with OA, which were significantly greater than the responses seen in strips from vehicle-challenged animals. In contrast, neither CPA, CGS 21580 nor 2-Cl-IB-MECA, applied at concentrations 100 times their Ki values for the rat A1, A2A and A3 receptors, respectively (Jacobson et al., 1995), induced contraction of the strips (Figure 8). When applied to the tissue, 15 min prior to challenge with adenosine (1 mM), neither 8-SPT (100 μM) nor MRS 1754 (1 μM) had any inhibitory effect on the response (Figure 9). In contrast, the response to adenosine was blocked significantly by CGS 15943 (1 μM). Reference to the dose-response curve to adenosine in Figure 9, indicates that the degree of inhibition was 3 fold from which a KB of 506 nM can be calculated.

Figure 8.

Figure 8

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Effects of adenosine, NECA, CPA, CGS 21680 and 2-Cl-IB-MECA on parenchymal strips prepared from lungs removed from Brown Norway rats actively sensitized to ovalbumin (OA) and challenged 3 h previously with vehicle (saline, 0.2 ml, i.t.) or OA (0.3 mg kg−1 i.t.). Responses are expressed relative to the response to 5-HT (10−4 M). Results are expressed as means±s.e.means of between three and six individual experiments. *P<0.05, **P<0.01 that the value is significantly different from the equivalent value in the vehicle-challenged group.

Figure 9.

Figure 9

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The effect of the adenosine receptor antagonists 8-SPT (100 μM), CGS 15943 (1 μM) and MRS 1754 (1 μM) on responses to adenosine (1 mM) on parenchymal strips prepared from lungs removed from Brown Norway rats actively sensitized to OA and challenged 3 h previously with OA (0.3 mg kg−1 i.t.). Responses are expressed relative to the maximal response to 5-HT (10−4 M). The results represent the means±s.e.means of 4 – 9 individual experiments. **P<0.01 that the value differs significantly from the response to adenosine (1 mM) in the absence of CGS 15943.

Discussion

We have used a range of adenosine receptor agonists and antagonists in order to define the adenosine receptor subtype which mediates the augmented bronchoconstrictor response to adenosine in actively sensitized, OA-challenged animals. With respect to the agonists, these were NECA, the archetypal broad-spectrum adenosine receptor agonist (Bruns et al., 1986), CPA, an A1-selective agonist (Londos et al., 1980), CGS 21680, an A2A-selective agonist (Jarvis et al., 1989; Wan et al., 1990), and 2-Cl-IB-MECA, an agonist with high affinity and selectivity for A3 adenosine receptors (Kim et al., 1994). With respect to the antagonists we used DPCPX, which is A1-selective (Bruns et al., 1987; Jacobson et al., 1999), ZM 241385, which is A2A-selective (Palmer et al., 1995; Poucher et al., 1995; Kim et al., 1998), MRS 1754, which is A2B -selective (Kim et al., 2000) and 8-SPT and CGS 15943, which are broad-spectrum adenosine receptor antagonists showing potency at A1, A2A, A2B receptors but negligible affinity for A3 receptors (Ji et al., 1994; Fozard & Hannon, 1999; see also Table 2). The data provide a pharmacological definition of the receptor subtype involved in the augmented bronchoconstrictor response to adenosine in actively sensitized, OA-challenged animals.

First, the ability of NECA, a stable analogue of adenosine with negligible affinity for the adenosine transporter (Balwierczak et al., 1989; Jimenez et al., 2000), to mimic the response to adenosine implies the involvement of cell surface adenosine receptors. Moreover, cross-desensitization readily occurs between adenosine and NECA (Fozard & Hannon, 2000) suggesting that the two agonists share a common mechanism of action. However, since NECA, like adenosine has affinity for each of the recognized adenosine receptor subtypes (Klotz et al., 1998; Ralevic & Burnstock, 1998), the observation provides no help in identifying which receptor site is involved. The analysis was therefore extended to include agonists selective for the adenosine A1, A2A and A3 receptors (CPA, CGS 21680 and 2-Cl-IB-MECA, respectively). The doses of CPA and CGS 21680 used in this study stem from the observations of Fozard & Carruthers (1993) who demonstrated, in pithed Sprague-Dawley rats with blood pressures supported by angiotensin II, pronounced bradycardic or hypotensive responses via the activation of A1 or A2A adenosine receptors, respectively. The doses of 2-Cl-IB-MECA were selected based on the observations of van schaick et al. (1996) who showed that intravenous injection of 200 μg kg−1 in the conscious Wistar rat induced hypotension and a marked increase in the plasma histamine concentration, a consequence of A3 receptor activation. The cardiovascular effects of CPA, CGS 21680 and 2-Cl-IB-MECA were confirmed in the present studies (Table 1), thus establishing that the doses used were sufficient for subtype-selective activation of the respective adenosine receptors.

The fact that at the 10 μg kg−1 dose of CPA and following 30 μg kg−1 CGS 21680, there was no bronchoconstriction despite clear cardiovascular evidence of A1 and A2A receptor activation, respectively, effectively rules out a role for these receptor subtypes in the bronchoconstrictor response to adenosine. However, small but significant bronchoconstrictor effects were seen following CPA (30 μg kg−1 i.v.) and the highest doses of 2-Cl-IB-MECA. Since such doses when administered to Sprague-Dawley or Wistar rats stimulate A3 receptors leading to mast cell degranulation (Fozard & Carruthers, 1993; Hannon et al., 1995, Fozard et al., 1996; van schaick et al., 1996), it is possible that the bronchoconstrictor effects of the higher doses of CPA and 2-Cl-IB-MECA in the present study reflect A3 receptor activation. It is important to emphasize however that the bronchoconstrictor responses to CPA and 2-Cl-IB-MECA are small (⩽100% increase over baseline) relative to those produced by adenosine or NECA (increases >850 % of basal values).

Thus, from the analysis of the effects of the adenosine receptor agonists, neither A1 nor A2A receptors appear to be involved in the bronchoconstrictor response to adenosine and the consequences of A3 receptor activation are minimal. The fact that the broad-spectrum adenosine receptor agonists, adenosine and NECA are effective suggests the A2B receptor as the major contributor to the bronchoconstrictor response to adenosine augmented following allergen challenge in the actively sensitized BN rat. We carried out an antagonist analysis to seek support for this conclusion.

DPCPX did not block the bronchoconstrictor response to adenosine despite strongly inhibiting the bradycardia induced by CPA. Similarly, ZM 241385 induced no blockade of the response to adenosine, despite markedly inhibiting the hypotensive response to CGS 21680. Bronchoconstrictor responses to 5-HT, used as a control for the sensitivity of the bronchial smooth muscle to the mediator involved in the response to adenosine (Hannon et al., 2001), were unaltered (DPCPX) or slightly reduced (ZM 241385). Together with the observations that neither CPA nor CGS 21680 induced bronchoconstriction, these data rule out unequivocally the involvement of A1 or A2A receptors in the bronchoconstrictor response to adenosine, augmented following OA-challenge in actively sensitized BN rats.

8-SPT induced dose-dependent blockade of the bronchoconstrictor response to adenosine with no effects on those to 5-HT. 8-SPT also inhibited the adenosine-induced increase in plasma histamine and 5-HT concentrations at the same dose at which the bronchoconstrictor effects are inhibited. These observations are consistent with the anti-bronchoconstrictor effect of 8-SPT being a consequence of blockade of the receptor present on airway mast cells through which adenosine induces mediator release and bronchoconstriction (Hannon et al., 2001). 8-SPT has broadly similar antagonist potencies at A1, A2A and A2B receptors but has only weak effects at rat A3 receptors (Ji et al., 1994; van galen et al., 1994; see also Table 2). Indeed, our earlier studies established that the higher dose of 8-SPT used in the present study (40 mg kg−1) did not block A3 receptor mediated hypotension in the rat (Fozard & Carruthers, 1993). Given that A1 and A2A receptors are not involved in the response to adenosine, the most plausible explanation for the marked and dose-dependent inhibition seen in animals treated with 8-SPT would be blockade of adenosine A2B receptors. CGS 15943, like 8-SPT, has affinity for rat A1, A2A and A2B receptors (although it is significantly more potent) and has negligible affinity for rat A3 receptors (Kim et al., 1998; Gao et al., 2001). Blockade of the response to adenosine by relatively low doses of CGS 15943 is again ostensibly in support of a role for A2B receptors.

Further support for this conclusion was sought by using MRS 1754, a xanthine derivative with high affinity for the human A2B receptor (Ki 2 nM) and selectivity (>200 fold) vis à vis the other human adenosine receptor subtypes (Kim et al., 2000). Although our data indicate that the KB for the rat A2B receptor is somewhat lower (16 nM) than that at the human receptor and similar to the Ki at the rat A1 receptor (17 nM; Kim et al., 2000), MRS 1754 provides a useful tool to implicate A2B receptors in the response. Surprisingly, MRS 1754 failed to block the bronchoconstrictor response to adenosine despite, at the higher dose of 3 mg kg−1, producing blockade of the A1 receptor-mediated bradycardia, induced by NECA. Given that MRS 1754 has approximately equal affinity for the rat A1 and A2B receptors and that the plasma concentration at the time of administration of adenosine is appreciably greater than that at the time of starting the NECA sequence (7 fold in the case of the 1 mg kg−1 dose level), the observation is difficult to reconcile with the involvement of A2B receptors in the bronchoconstrictor response to adenosine.

Consideration of the pharmacokinetic profile of 8-SPT after intravenous administration under identical conditions to those of the functional studies further questions the role of the A2B receptor in the bronchoconstrictor response to adenosine augmented after allergen challenge. Thus, following a dose of 20 mg kg−1 of 8-SPT, which induced an approximate 3 fold blockade of the bronchoconstrictor response to adenosine, the concentration of 8-SPT present in the plasma at the time of adenosine challenge was 139 fold higher than its KB at the rat A2B receptor. This should be contrasted with the substantially greater than 3 fold blockade of the A1 receptor-mediated bradycardia induced by NECA between 30 and 40 min after drug administration when the plasma concentration exceeded the KB at the rat A1 receptor by only 6.6 fold. Similar reasoning can be applied to CGS 15943, where the plasma concentrations at the times of testing adenosine and NECA exceed the KB values at the A2B and A1 receptors by 21 and 16 fold, respectively, and the degree of blockade of the A1 receptor-mediated bradycardia is clearly greater than that of the bronchoconstrictor response to adenosine. Although a possible confounding factor in relating plasma drug concentrations to pharmacodynamic responses is the degree of protein binding, this parameter is independent of drug concentration and would not detract from the reasoning set out above.

Finally, data from our in vitro studies lend strong support to the conclusion that the augmented response to adenosine following allergen challenge is not mediated by A2B receptors. The augmented contractile response of lung parenchymal strips removed from actively sensitized OA-challenged animals to adenosine mirrors closely the in vivo bronchoconstrictor response with respect to agonist pharmacology (Figures 1 and 8) and the involvement of mast cells (Hannon et al., 2001). Nevertheless, the response to adenosine is resistant to blockade by 8-SPT, and MRS 1754 at concentrations >50 times their respective KB values for blockade of the rat A2B receptor. Moreover, although CGS 15943 is able to antagonize the response to adenosine, its affinity in this respect (KB, 506 nM) is appreciably less than its affinity for the A2B receptor of the longitudinal muscle of the rat colon (KB 70 nM) defined under similar experimental conditions.

Thus, we are left with the surprising conclusion that the receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in the actively sensitized BN rat cannot be categorized as one of the four recognized adenosine receptor subtypes. It remains a challenge for the future to define this receptor further and to establish whether the receptor mediating the response to adenosine of the human asthmatic airways is the same.

Acknowledgments

We thank Dr Roger Taylor for the synthesis of MRS 1754, and Elisabeth Schaeublin and Francois Baur for their expert technical assistance.

Abbreviations

AHR

airway hyperresponsiveness

BN

Brown Norway

CGS 21680

2-[p-(2-carboxylethyl) phenethylamino]-5′-N-ethylcarboxamidoadenosine

2-Cl-IB-MECA

2-chloro-N6-(3-iodobenzyl) 5′-N-methylcarboxamidoadenosine

CGS 15943

9-chloro-2,2-(furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine

CPA

N6-cyclopentyladenosine

DMSO

dimethylsulphoxide

DPCPX

1,3-dipropyl-8-cyclopentylxanthine

HR

heart rate

5-HT

5-hydroxytryptamine

i.t.

intratracheal

LC-MS/MS

Liquid chromatography-mass spectrometry/mass spectrometry

MABP

mean arterial blood pressure

MRS 1754

N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide

NECA

5′-N-ethylcarboxamido adenosine

OA

ovalbumin

8-SPT

8-(p-sulphophenyl) theophylline

ZM 241385

4-(2-[7-amino-2-(2-furyl) [1,2,4] triazolo [2,3-a][1,3,5] triazin-5-yl amino]ethyl) phenol

References

  1. BAILEY S.J., HOURANI S.M.O. Effects of purines on the longitudinal muscle of the rat colon. Br. J. Pharmacol. 1992;105:885–892. doi: 10.1111/j.1476-5381.1992.tb09073.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BALWIERCZAK J.L., KRULAN C.M., WANG Z.C., CHEN J., JENG A.Y. Effects of adenosine A2 receptor agonists on nucleoside transport. J. Pharmacol. Exp. Ther. 1989;251:279–287. [PubMed] [Google Scholar]
  3. BRUNS R.F., FERGUS J.H., BADGER E.W., BRISTOL J.A., SANTAY L.A., HARTMAN J.D., HAYS S.J., HUANG C.C. Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1-3-dipropylxanthine to rat brain membranes. Naunyn-Schmiedeberg's Arch. Pharmacol. 1987;335:59–63. doi: 10.1007/BF00165037. [DOI] [PubMed] [Google Scholar]
  4. BRUNS R.F., LU G.H., PUGSLEY G.H. Characterisation of the A2 adenosine receptor labelled by [3H]NECA in rat striatal membranes. Mol. Pharmacol. 1986;29:331–346. [PubMed] [Google Scholar]
  5. FEOKTISTOV I., POLOSA R., HOLGATE S.T., BIAGGIONI I. Adenosine A2B receptors: a novel therapeutic target in asthma. Trends Pharmacol. Sci. 1998;19:148–153. doi: 10.1016/s0165-6147(98)01179-1. [DOI] [PubMed] [Google Scholar]
  6. FORSYTHE P., ENNIS M. Adenosine, mast cells and asthma. Inflamm. Res. 1999;48:301–307. doi: 10.1007/s000110050464. [DOI] [PubMed] [Google Scholar]
  7. FOZARD J.R., CARRUTHERS A.M. The cardiovascular effects of selective adenosine A1 and A2 receptor agonists in the pithed rat: no role for glibenclamide-sensitive potassium channels. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993;347:192–196. doi: 10.1007/BF00169266. [DOI] [PubMed] [Google Scholar]
  8. FOZARD J.R., HANNON J.P. Adenosine receptor ligands: potential as therapeutic agents in asthma and COPD. Pulm. Pharmacol. Ther. 1999;12:111–114. doi: 10.1006/pupt.1999.0191. [DOI] [PubMed] [Google Scholar]
  9. FOZARD J.R., HANNON J.P. Species differences in adenosine induced bronchoconstrictor responses. Clin. Exp. Allergy. 2000;30:1213–1220. doi: 10.1046/j.1365-2222.2000.00894.x. [DOI] [PubMed] [Google Scholar]
  10. FOZARD J.R., PFANNKUCHE H.-J., SCHUURMANN H.-J. Mast cell degranulation following adenosine A3 receptor activation in rats. Eur. J. Pharmacol. 1996;298:293–297. doi: 10.1016/0014-2999(95)00822-5. [DOI] [PubMed] [Google Scholar]
  11. FOZARD J.R., TIGANI B., HANNON J.P. Hyperresponsiveness of lung parenchymal strips to adenosine receptor agonists induced by antigen challenge in actively sensitised Brown Norway (BN) rats. Br. J. Pharmacol. 2001;133:42P. doi: 10.1038/sj.bjp.0703961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. FURCHGOTT R.F.The classification of adrenoceptors (adrenergic receptors). An evaluation from the stand point of receptor theory Catecholamines 1972Heidelberg: Springer-Verlag; 283–335.eds. Blaschko, H. & Muscholl, E. pp [Google Scholar]
  13. GAO Z., LI B.-S., DAY Y.-J., LINDEN J. A3 adenosine receptor activation triggers phosphorylation of protein kinase B and protects rat basophilic leukaemia 2H3 mast cells from apoptosis. Mol. Pharmacol. 2001;59:76–82. doi: 10.1124/mol.59.1.76. [DOI] [PubMed] [Google Scholar]
  14. HANNON J.P., PFANNKUCHE H.-J., FOZARD J.R. A role for mast cells in adenosine A3 receptor-mediated hypotension in the anaesthetised rat. Br. J. Pharmacol. 1995;115:945–952. doi: 10.1111/j.1476-5381.1995.tb15902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. HANNON J.P., TIGANI B., MAZZONI L., FOZARD J. R. Mechanism of airway hyperreactivity to adenosine induced by allergen challenge in actively sensitised Brown Norway rats. Br. J. Pharmacol. 1999a;127:76P. doi: 10.1038/sj.bjp.0703961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. HANNON J.P., TIGANI B., MAZZONI L., FOZARD J. R. The receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitised Brown Norway rats. Br. J. Pharmacol. 1999b;127:77P. doi: 10.1038/sj.bjp.0704516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. HANNON J.P., TIGANI B., WILLIAMS I., MAZZONI L., FOZARD J.R. Mechanism of airway hyperresponsiveness to adenosine induced by allergen challenge in actively sensitised Brown Norway rats. Br. J. Pharmacol. 2001;132:1509–1523. doi: 10.1038/sj.bjp.0703961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. JACOBSON M.A., BAI T.R.The role of adenosine in asthma Purinergic approaches in experimental therapeutics 1997Wiley-Liss; 315–331.eds Jacobson, K.A. & Jarvis, M.F. pp [Google Scholar]
  19. JACOBSON K.A., IJZERMAN A.P., LINDEN J. 1,3-Dialkylxanthine derivative having high potency as antagonists at human A2B adenosine receptors. Drug Dev. Res. 1999;47:45–53. doi: 10.1002/(sici)1098-2299(199905)47:1&#x0003c;45::aid-ddr6&#x0003e;3.0.co;2-u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. JACOBSON K.A., KIM H.O., SIDDIQI S.M., OLAH M.E., STILES G.L., VON LUBITZ D.KG.E. A3-Adenosine receptors: design of selective ligands and therapeutic prospects. Drugs of the Future. 1995;20:689–699. doi: 10.1358/dof.1995.020.07.531583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. JARVIS M.F., SCHULTZ R., HUTCHISON A.J., HOI-DU U., SILLS M.A., WILLIAMS M. [3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain. J. Pharm. Exp. Thera. 1989;251:888–893. [PubMed] [Google Scholar]
  22. JI X.-D., VON LUBITZ D., OLAH M.E., STILES G.L., JACOBSON K.A. Species differences in ligand affinity at central A3 adenosine receptors. Drug Dev. Res. 1994;33:51–59. doi: 10.1002/ddr.430330109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. JIMENEZ A., PUBILL D., PALLAS M., CAMINS A., LLADO S., CAMARASA J., ESCUBEDO E. Further characterisation of an adenosine transport system in the mitochondrial fraction of rat testis. Eur. J. Pharmacol. 2000;398:31–39. doi: 10.1016/s0014-2999(00)00297-1. [DOI] [PubMed] [Google Scholar]
  24. KIM H.O., JI X.-D., SIDDIQI S.M., OLAH M.E., STILES G.L., JACOBSON K.A. 2-Substitution of N6-benzyladenosine-5′-uronamides enhances selectivity for A3 adenosine receptors. J. Med. Chem. 1994;37:3614–3621. doi: 10.1021/jm00047a018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. KIM Y.C., DE ZWART M., CHANG L., MORO S., VON FRIJTAG DRABBE KUENZEL J.K., MELMAN N., IJZERMAN A.P., JACOBSON K.A. Derivatives of the triazoloquinazoline adenosine antagonist having high potency at the human A2B and A3 receptor subtypes. J. Med. Chem. 1998;41:2835–2845. doi: 10.1021/jm980094b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. KIM Y.C., JI X.D., MELMAN N., LINDEN J., JACOBSON K.A. Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at human A2B adenosine receptors. J. Med. Chem. 2000;43:1165–1172. doi: 10.1021/jm990421v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. KLOTZ K.N., HESSLING J., HEGLER J., OWMAN C., KULL B., FREDHOLM B.B., LOHSE M.J. Comparative pharmacology of human adenosine receptor subtypes - characterisation of stably transfected receptors in CHO cells. Naunyn Schmiedeberg's Arch. Pharmacol. 1998;357:1–9. doi: 10.1007/pl00005131. [DOI] [PubMed] [Google Scholar]
  28. LONDOS C., COOPER D.M.F., WOLFF J. Subclasses of external adenosine receptors. Proc. Natl. Acad. Sci. U.S.A. 1980;77:2551–2554. doi: 10.1073/pnas.77.5.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. MARQUARDT D.L.Adenosine Asthma 1997Lippencourt-Raven; 585–591.eds Barnes, P.J., Grunstein, M.M., Leff, A.R., Woolcock, A.J. pp [Google Scholar]
  30. MEADE C.J., DUMONT I., WORRALL L. Why do asthmatic subjects respond so strongly to inhaled adenosine. Life Sci. 2001;69:1225–1240. doi: 10.1016/s0024-3205(01)01231-0. [DOI] [PubMed] [Google Scholar]
  31. PALMER T.M., POUCHER S.M., JACOBSON K.A., STILES G.L. 125I-4-(2-[7-Amino-2{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl) phenol, a high affinity antagonist radioligand selective for the A2A adenosine receptor. Mol. Pharmacol. 1995;48:970–974. [PMC free article] [PubMed] [Google Scholar]
  32. PALSER A., HANNON J.P., TIGANI B., MAZZONI L., FOZARD J.R. Time dependent changes in bronchial responsiveness to direct and indirect bronchospasmogens following allergen challenge in actively sensitised Brown Norway rats. Br. J. Pharmacol. 2000;131:84P. [Google Scholar]
  33. PHILLIPS G.D., HOLGATE S.T.Adenosine and the airways in asthma Asthma and Rhinitis 1995Blackwell Scientific; 851–857.eds Busse, W.W., Holgate, S.T. pp [Google Scholar]
  34. POLOSA R., HOLGATE S.T. Adenosine bronchoprovocation: a promising marker of allergic inflammation in asthma. Thorax. 1997;52:919–923. doi: 10.1136/thx.52.10.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. POUCHER S.M., KEDDIE J.R., SINGH P., STOGGALL S.M., CAULKETT P.W.R., JONES G., COLLIS M.G. The in vitro pharmacology of ZM 241385, a potent, non-xanthine, A2A selective adenosine receptor antagonist. Br. J. Pharmacol. 1995;115:1096–1102. doi: 10.1111/j.1476-5381.1995.tb15923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. RALEVIC V., BURNSTOCK G. Receptors for purines and pyrimidines. Pharmacol. Rev. 1998;50:413–492. [PubMed] [Google Scholar]
  37. TARAYRE J.P., ALIAGA M., BARBARA M., TISSEYRE N., VIEU S., TISNE-VERSAILLES J. Model of bronchial allergic inflammation in the Brown-Norway rat. Pharmacological modulation. Int. J. Immunopharmacol. 1992;14:847–855. doi: 10.1016/0192-0561(92)90083-w. [DOI] [PubMed] [Google Scholar]
  38. TIGANI B., HANNON J.P., MAZZONI L., FOZARD J.R. Effects of wortmannin on bronchoconstrictor responses to adenosine in actively sensitised Brown Norway rats. Eur. J. Pharmacol. 2000;406:469–476. doi: 10.1016/s0014-2999(00)00705-6. [DOI] [PubMed] [Google Scholar]
  39. VAN GALEN P.JM., VAN BERGEN A.H., GALLO-RODRIGUEZ C., MELMAN N., OLAH M.E., IJZERMAN A.P., STILES G.L., JACOBSON K.A. A binding site model and structure-activity relationships for the rat A3 adenosine receptor. Mol. Pharmacol. 1994;45:1101–1111. [PMC free article] [PubMed] [Google Scholar]
  40. VAN SCHAICK E.A., JACOBSON K.A., KIM H.O., IJZERMAN A.P., DANHOF M. Haemodynamic effects and histamine release elicited by the selective adenosine A3 receptor agonist 2-Cl-IB-MECA in conscious rats. Eur. J. Pharmacol. 1996;308:311–314. doi: 10.1016/0014-2999(96)00373-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. WAN W., SUTHERLAND G.R., GEIGER J.D. Binding of the adenosine A2 receptor ligand [3H]CGS 21680 to human and rat brain: evidence for multiple affinity sites. J. Neurochem. 1990;55:1763–1771. doi: 10.1111/j.1471-4159.1990.tb04967.x. [DOI] [PubMed] [Google Scholar]

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