Short communicationComparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells1
Introduction
Most of the effects of the most widely used of all psychoactive drugs, caffeine, are believed to be secondary to the blockade of adenosine receptors [1]. Since caffeine is an antagonist, this implies that at least some adenosine receptors are occupied in vivo by the endogenous ligand. Four adenosine receptors, A1, A2A, A2B, and A3, have been cloned and pharmacologically characterized from several mammalian species [2]. It is generally assumed that adenosine is the natural ligand at all these receptors, but it was recently suggested that inosine may be a more important agonist than adenosine at the A3 receptors [3]. It is also generally believed that adenosine is a much more potent agonist at A1 and A2A receptors than at A2B and A3 receptors [see e.g. Ref. 4]. However, this contention is largely based on studies on cells with widely different levels of receptor expression, and no direct comparison has been made. In addition, most studies on the potency of agonists at adenosine receptors are performed using binding assays. It is notoriously difficult to study the potency of adenosine itself in such a preparation [5], [6], [7], because adenosine is present in most membrane preparations and binds with very high affinity to the A1 receptor in its high-affinity conformation in the absence of guanine nucleotides [8], [9], [10].
In the present series of experiments, we therefore examined the potency of adenosine and inosine to alter cAMP levels (increases in the case of A2A and A2B receptors; decreases in the case of A1 or A3 receptors) using intact CHO cells that express the human forms of the receptor. In addition, the potency of caffeine, theophylline, paraxanthine, and theobromine as antagonists was determined.
Section snippets
Materials
Forskolin, NBMPR, adenosine deaminase, caffeine, theophylline, and paraxanthine were from Sigma. Adenosine was from Aldrich-Europe and inosine from P-L Biochemicals. NECA and N6-cyclopentyl adenosine (CPA) were from RBI. Rolipram (ZK 62711) was a gift from Schering A. Cell culture medium and additions were from GIBCO.
Cell culture
CHO cells transfected with the human forms of the adenosine A1, A2B and A3[11] and A2A[12] receptors were cultured as described in these papers. Briefly, the cells were grown
Results
To examine the potency of adenosine as a receptor agonist at human adenosine receptors, we used changes in cAMP formation as a functional assay. In the case of A2A and A2B receptors this assay is straightforward, since agonists increase cAMP formation. However, both A1 and A3 receptors cause a decrease in the rate of cAMP formation. Therefore, a means to increase the rate of cAMP formation was needed. We chose forskolin as the agent to achieve this increase because in lower doses forskolin is
Discussion
Adenosine was, as expected, a full agonist at all four human adenosine receptors. The highest potency was observed at the A1 and A3 receptors, followed by the A2A receptor. Adenosine was much less potent at the A2B receptor. This study appears to be the first where the potency of adenosine as an agonist at all the human receptors has been directly compared. Using a luciferase construct under the control of multiple cyclic AMP response elements, an earlier study reported the effects of adenosine
Acknowledgements
These studies were supported by the Swedish Medical Research Council (Proj. No. 2553) and by the European Commission (EURCAR).
References (28)
Adenosine deaminase for removing adenosinehow much is enough?
Trends Pharmacol Sci
(1989)- et al.
The affinity of adenosine for the high- and low-affinity states of the human adenosine A1 receptor
Eur J Pharmacol
(1996) - et al.
Differences in the order of potency for agonists, but not antagonists, at human and rat adenosine A2A receptors
Biochem Pharmacol
(1999) - et al.
A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid
Anal Biochem
(1990) - et al.
Purine levels in the intact rat brain. Studies with an implanted perfused hollow fibre
Neurosci Lett
(1982) - et al.
Protective effect of adenosine and a novel xanthine derivative propentofylline on the cell damage after bilateral carotid occlusion in the gerbil hippocampus
Brain Res
(1990) - et al.
Reciprocal interactions between adenosine A2A and dopamine D2 receptors in Chinese hamster ovary cells co-transfected with the two receptors
Biochem Pharmacol
(1999) - et al.
Adenosine transport in nervous system tissues
- et al.
Adenosine transporters
Gen Pharmacol
(1996) - et al.
The use of caffeine as a metabolic probe for human drug metabolizing enzymes
Gen Pharmacol
(1996)
Actions of caffeine in the brain with special reference to factors that contribute to its widespread use
Pharmacol Rev
Nomenclature and classification of purinoceptors
Pharmacol Rev
Inosine binds to A3 adenosine receptors and stimulates mast cell degranulation
J Clin Invest
Adenosine, adenosine receptors and the actions of caffeine
Pharmacol Toxicol
Cited by (377)
Nucleoside transporters and immunosuppressive adenosine signaling in the tumor microenvironment: Potential therapeutic opportunities
2022, Pharmacology and TherapeuticsLuciferase-based GloSensor™ cAMP assay: Temperature optimization and application to cell-based kinetic studies
2022, MethodsCitation Excerpt :Cells were stimulated with various concentrations of adenosine, and maximal slopes were used to construct concentration–response curves (e.g., Fig. 3B). In this case, adenosine yielded a sigmoidal concentration dependence curve with pEC50 of 5.7, in keeping with previous reports for the adenosine A2a receptor [35]. HEK293H cells co-transfected with the pGloSensor-22F cAMP plasmid and adenosine A2a receptor plasmid were incubated with D-luciferin and IBMX at room temperature.
Engineering CREB-activated promoters for adenosine-induced gene expression
2024, Biotechnology JournalExtracellular Purine Metabolism—Potential Target in Multiple Sclerosis
2024, Molecular Neurobiology
- 1
Abbreviations: cAMP, cyclic adenosine 3′,5′-monophosphate; CHO, Chinese hamster ovary; NBMPR, nitrobenzylthioinosine; and NECA, 5′-N-ethyl carboxamido adenosine.