Pharmacological characterization of adenosine A2B receptors: Studies in human mast cells co-expressing A2A and A2B adenosine receptor subtypes

doi:10.1016/S0006-2952(97)00512-1

Biochemical Pharmacology

Volume 55, Issue 5, 1 March 1998, Pages 627-633

https://doi.org/10.1016/S0006-2952(97)00512-1 Get rights and content

Abstract

Characterization of A_2B receptors is hampered by the lack of selective pharmacological probes and often relies on their relative affinity to agonists that are selective at other receptor types. This approach is limited because the affinity of A_2B receptors for putative A₃ agonists has not been determined. Using the human erythroleukemia cell line HEL as a cellular model for A_2B-mediated adenylate cyclase activation, we found the following potencies (pD₂) for the non-selective agonist 5′-N-ethylcarboxamidoadenosine (NECA) (5.65 ± 0.04), the putative A₃ agonists N⁶-benzyl-NECA (4.17 ± 0.06) and N⁶-(3-iodobenzyl)-N-methyl-5′-carbamoyl-adenosine (IB-MECA) (3.7 ± 0.02), and the A_2A agonist 4-[(N-ethyl-5′-carbamoyladenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS21680) (2.8 ± 0.1). Because of the lack of a selective agonist, characterization of A_2B receptor function is difficult in cells co-expressing A_2A receptors. In the human mast cell line HMC-1, NECA induced cAMP accumulation with a concentration-response relationship best fitted to a two-sited model (pD₂ 7.69 ± 0.42 and 5.92 ± 0.21 for high- and low-affinity sites), suggesting the presence of both A_2A and A_2B receptors in these cells. We demonstrated that A_2B receptors can be selectively activated with NECA in the presence of the selective A_2A antagonist 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261). Under these conditions, the concentration-response relationship of NECA for cyclic AMP accumulation was now best fitted to a one-site model (pD₂ 5.68 ± 0.03, Hill slope 0.93 ± 0.06, 95% confidence intervals 0.8 to 1.06)corresponding to selective activation of A_2B receptors. Using the approaches developed in this study, we determined that A_2B, and not A_2A or A₃, receptors account for all the calcium mobilization induced by NECA in HMC-1 cells.

References (34)

PL Martin
Relative agonist potencies of C²-substituted analogues of adenosine: Evidence for adenosine A_2B receptors in the guinea pig aorta
Eur J Pharmacol
(1992)
T Iwamoto et al.
Identification of adenosine A₂ receptor-cAMP System in human aortic endothelial cells
Biochem Biophys Res Commun
(1994)
GR Strohmeier et al.
The A_2b adenosine receptor mediates cAMP responses to adeno-sine receptor agonists in human intestinal epithelia
J Biol Chem
(1995)
A Gharib et al.
Evidence for adenosine A_2b receptors in the rat pineal gland
Eur J Pharmacol
(1992)
M Okada et al.
Adenosine A₁ and A₂ receptors modulate extracellular dopamine levels in rat striatum
Neurosci Lett
(1996)
G Grynkiewicz et al.
A new generation of Ca²⁺ indicators with greatly improved fluorescence properties
J Biol Chem
(1985)
V Ramkumar et al.
The A₃ adenosine receptor is the unique adenosine receptor which facilitates release of allergic mediators in mast cells
J Biol Chem
(1993)
JW Daly et al.
Subclasses of adenosine receptors in the central nervous system: Interaction with caffeine and related methylxanthines
Cell Mol Neurobiol
(1983)
RF Bruns et al.
Characterization of the A₂ adenosine receptor labeled by [³H]NECA in rat striatal membranes
Mol Pharmacol
(1986)
I Feoktistov et al.
Adenosine A_2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma
J Clin Invest
(1995)

DL Marquardt et al.

Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells

J Immunol

(1994)

DL Boyle et al.

Inhibition of synoviocyte collagenase gene expression by adenosine receptor stimulation

Arthritis Rheum

(1996)

BL Fiebich et al.

Adenosine A_2b receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells

J Neurochem

(1996)

RK Dubey et al.

Adenosine inhibits growth of rat aortic smooth muscle cells. Possible role of A_2b receptor

Hypertension

(1996)

PL Martin et al.

2-Phenylethoxy-9-methyladenine: An adenosine receptor antagonist that discriminates between A₂ adenosine receptors in the aorta and the coronary vessels from the guinea pig

J Pharmacol Exp Ther

(1993)

A Rubino et al.

Contribution of P₁-(A_2b subtype) and P₂-purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed

Br J Pharmacol

(1995)

JJ Haynes et al.

Ade-nosine-induced vasodilation: Receptor characterization in pulmonary circulation

Am J Physiol

(1995)

Cited by (79)

Characterisation of endogenous A<inf>2A</inf> and A<inf>2B</inf> receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A<inf>2B</inf>-selective antagonist PSB 603
2018, Biochemical Pharmacology
Citation Excerpt :
Interestingly, BAY 60-6583 gave a similar time course (providing evidence of desensitization at higher agonist concentrations) to that observed with NECA (Fig. 2c) whilst CGS 21680 did not (Fig. 2d). In addition the concentration-response curve for NECA had a Hill slope significantly less than unity (0.75; p < .001; Partial F test) which would be consistent with the presence of more than one component, similar to that seen previously in HMC-1 cells, which also expresses both A2AAR and A2BAR subtypes [32]. The partial agonist nature of the response to the A2B-selective agonist BAY 60-6583 at the A2BAR was confirmed in combination experiments with NECA (Fig. 5a).
Endogenous adenosine A_2B receptors (A_2BAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A_2BAR antagonist PSB 603 in HEK 293 cells. The A_2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A_2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A_2AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E_MAX with no significant effect on EC₅₀ values. Kinetics analysis of the effect of PSB 603 on the A_2BAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A_2BAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.
Theophylline
2009, Asthma and COPD
Theophylline remains one of the most widely prescribed drugs for the treatment of asthma and chronic obstructive pulmonary disease (COPD) worldwide, since it is inexpensive and widely available. The frequency of side-effects at the previously recommended doses and the relatively low efficacy of theophylline have recently led to reduced usage, since inhaled β₂-agonists are far more effective as bronchodilators and inhaled corticosteroids have a greater anti-inflammatory effect. Despite the fact that theophylline has been used in asthma therapy for over 70 years, there is still considerable uncertainty about its molecular mode of action in asthma and its logical place in therapy. Theophylline is a methylxanthine similar in structure to the common dietary xanthines caffeine and theobromine. Many salts of theophylline have also been marketed, the most common being aminophylline, the ethylene diamine salt used to increase solubility at neutral pH, so that intravenous administration is possible. Theophylline has several cellular effects that may contribute to its clinical efficacy in the treatment of asthma. The primary effect of theophylline is assumed to be the relaxation of airway smooth muscle. It is suggested that theophylline may exert its effects in asthma via some action outside the airways. It may be relevant that theophylline is ineffective when given by inhalation until therapeutic plasma concentrations are achieved. The molecular mechanism of anti-inflammatory effects of theophylline is now becoming clearer, and it seems likely that there is a synergistic interaction with the anti-inflammatory mechanism of corticosteroids through restoration of HDAC activity. This interaction may underlie the beneficial effects of theophylline when added to inhaled corticosteroids. This may be particularly appropriate in patients with more severe asthma in whom corticosteroids are less effective as there may be a reduction in HDAC activity in these patients as well as in smoking asthmatics patients and patients with COPD.
The RegEx trial: A randomized, double-blind, placebo- and active-controlled pilot study combining regadenoson, a selective A<inf>2A</inf> adenosine agonist, with low-level exercise, in patients undergoing myocardial perfusion imaging
2009, Journal of Nuclear Cardiology
Although vasodilator stress myocardial perfusion imaging (MPI) is increasingly performed with exercise, adenosine A_2A receptor agonists have not been studied with exercise.
To determine the safety of administering regadenoson during exercise and, secondarily, to evaluate image quality, patient acceptance, and detection of perfusion defects.
Patients requiring pharmacologic MPI received a standard adenosine-supine protocol (AdenoSup, n = 60) and were then randomized (2:1) in a double-blind manner to low-level exercise with bolus intravenous injection of regadenoson (RegEx, n = 39) or placebo (PlcEx, n = 21).
Adverse events occurred in 95%, 77%, and 33% of patients receiving AdenoSup, RegEx, and PlcEx, respectively. Peak heart rate was 13 beats per minute (bpm) and 21 bpm greater following RegEx compared to that following PlcEx and AdenoSup, respectively (P = .006 and <.001). Change from baseline in mean systolic blood pressure (SBP), change from baseline to nadir SBP, and percentage of patients with a decline in SBP by ≥20 mm Hg showed no important differences between RegEx and PlcEx. No occurrences of 2nd degree or higher AV block were observed following RegEx or PlcEx; one patient developed 2nd degree AV block following AdenoSup. The mean heart-to-liver and heart-to-gut ratios were improved on RegEx vs AdenoSup: 0.85 (0.34) vs 0.65 (0.26), P < .001 and 1.1 (0.36) vs 0.97 (0.34), P < .001, respectively. Compared to AdenoSup, 70% of patients felt RegEx was much or somewhat better.
Combining regadenoson with low-level exercise is feasible, well tolerated, and associated with fewer side effects compared to AdenoSup.
Theophylline
2008, Asthma and COPD: Basic Mechanisms and Clinical Management
Theophylline remains one of the most widely prescribed drugs for the treatment of asthma and chronic obstructive pulmonary disease (COPD) worldwide, since it is inexpensive and widely available. The frequency of side-effects at the previously recommended doses and the relatively low efficacy of theophylline have recently led to reduced usage, since inhaled β₂-agonists are far more effective as bronchodilators and inhaled corticosteroids have a greater anti-inflammatory effect. Despite the fact that theophylline has been used in asthma therapy for over 70 years, there is still considerable uncertainty about its molecular mode of action in asthma and its logical place in therapy. Theophylline is a methylxanthine similar in structure to the common dietary xanthines caffeine and theobromine. Many salts of theophylline have also been marketed, the most common being aminophylline, the ethylene diamine salt used to increase solubility at neutral pH, so that intravenous administration is possible. Theophylline has several cellular effects that may contribute to its clinical efficacy in the treatment of asthma. The primary effect of theophylline is assumed to be the relaxation of airway smooth muscle. It is suggested that theophylline may exert its effects in asthma via some action outside the airways. It may be relevant that theophylline is ineffective when given by inhalation until therapeutic plasma concentrations are achieved. The molecular mechanism of anti-inflammatory effects of theophylline is now becoming clearer, and it seems likely that there is a synergistic interaction with the anti-inflammatory mechanism of corticosteroids through restoration of HDAC activity. This interaction may underlie the beneficial effects of theophylline when added to inhaled corticosteroids. This may be particularly appropriate in patients with more severe asthma in whom corticosteroids are less effective as there may be a reduction in HDAC activity in these patients as well as in smoking asthmatics patients and patients with COPD.
Host A<inf>2B</inf> receptors promote carcinoma growth
2008, Neoplasia
Recent studies suggest that tumor-infiltrating immune cells can benefit the tumor by producing factors that promote angiogenesis and suppress immunity. Because the tumor microenvironment is characterized by high adenosine levels, we hypothesized that the low-affinity A_2B adenosine receptor located on host immune cells may participate in these effects. In the current study, we tested this hypothesis in a Lewis lung carcinoma isograft model using A_2B receptor knockout (A_2BKO) mice. These mice exhibited significantly attenuated tumor growth and longer survival times after inoculation with Lewis lung carcinoma compared to wild type (WT) controls. Lewis lung carcinoma tumors in A_2BKO mice contained significantly lower levels of vascular endothelial growth factor (VEGF) compared to tumors growing in WT animals. This difference was due to VEGF production by host cells, which comprised 30 ± 2% of total tumor cell population. Stimulation of adenosine receptors on WT tumorinfiltrating CD45⁺ immune cells increased VEGF production fivefold, an effect not seen in tumor-associated CD45⁺ immune cells lacking A_2B receptors. In contrast, we found no significant difference in VEGF production between CD45⁻ tumor cells isolated from WT and A_2BKO mice. Thus, our data suggest that tumor cells promote their growth by exploiting A_2B adenosine receptor–dependent regulation of VEGF in host immune cells.
Effects of IL-4 and IL-13 on adenosine receptor expression and responsiveness of the human mast cell line 1
2008, International Immunopharmacology
Inhalation of adenosine-5′-monophosphate (AMP) causes bronchoconstriction in asthma but not in healthy subjects. Bronchoconstriction upon AMP inhalation is thought to occur by histamine release and subsequent binding to receptors on airway smooth muscle cells.
To explain enhanced sensitivity to AMP in asthma, mast cell expression of the adenosine A_2A and A_2B receptors and histamine release were measured after incubation of human mast cell line 1 (HMC-1) cells with AMP and the non-specific adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) for 1.5 and 6 h. To establish a Thelper-2 environment resembling the asthma phenotype, HMC-1 cells were additionally cultured with IL-4 and IL-13 alone or stimulated with the combination of both cytokines and AMP and NECA. To study effects of prolonged presence of the inflammatory environment, the cells were pre-incubated overnight (18 h) with IL-4 and IL-13 and additionally stimulated with AMP and NECA for 1.5 or 6 h.
AMP and NECA hardly affected adenosine receptor expression but increased IL-8 secretion. Incubation with IL-4 and IL-13 for 6 h increased adenosine A_2A receptor expression and histamine secretion, but decreased IL-8 secretion. The combination of IL-4, IL-13, and AMP/NECA for 6 h increased A_2B receptor expression and IL-8 secretion. Overnight stimulation with IL-4, IL-13 and subsequent stimulation with AMP/NECA for 1.5 h decreased A_2AR expression which was accompanied by increased histamine secretion.
These results suggest a role for decreased A_2AR expression in enhanced adenosine responsiveness as observed in asthma.

View all citing articles on Scopus

^☆: This work also was supported by NIH Grants R29HL55596 and RR00095.

View full text

Research paperPharmacological characterization of adenosine A2B receptors: Studies in human mast cells co-expressing A2A and A2B adenosine receptor subtypes☆

Abstract

Eur J Pharmacol

Biochem Biophys Res Commun

J Biol Chem

Eur J Pharmacol

Neurosci Lett

J Biol Chem

J Biol Chem

Subclasses of adenosine receptors in the central nervous system: Interaction with caffeine and related methylxanthines

Cell Mol Neurobiol

Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes

Mol Pharmacol

Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma

J Clin Invest

Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells

J Immunol

Inhibition of synoviocyte collagenase gene expression by adenosine receptor stimulation

Arthritis Rheum

Adenosine A2b receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells

J Neurochem

Adenosine inhibits growth of rat aortic smooth muscle cells. Possible role of A2b receptor

Hypertension

2-Phenylethoxy-9-methyladenine: An adenosine receptor antagonist that discriminates between A2 adenosine receptors in the aorta and the coronary vessels from the guinea pig

J Pharmacol Exp Ther

Contribution of P1-(A2b subtype) and P2-purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed

Br J Pharmacol

Ade-nosine-induced vasodilation: Receptor characterization in pulmonary circulation

Am J Physiol

Research paper
Pharmacological characterization of adenosine A_2B receptors: Studies in human mast cells co-expressing A_2A and A_2B adenosine receptor subtypes☆

Characterization of the A₂ adenosine receptor labeled by [³H]NECA in rat striatal membranes

Adenosine A_2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma

Adenosine A_2b receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells

Adenosine inhibits growth of rat aortic smooth muscle cells. Possible role of A_2b receptor

2-Phenylethoxy-9-methyladenine: An adenosine receptor antagonist that discriminates between A₂ adenosine receptors in the aorta and the coronary vessels from the guinea pig

Contribution of P₁-(A_2b subtype) and P₂-purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed