Elsevier

Cellular Signalling

Volume 28, Issue 6, June 2016, Pages 552-560
Cellular Signalling

The adenosine metabolite inosine is a functional agonist of the adenosine A2A receptor with a unique signaling bias

https://doi.org/10.1016/j.cellsig.2016.02.010Get rights and content

Highlights

  • Inosine directly and physically engages the A2AR.

  • Inosine dose-dependently stimulates cAMP production mediated through the A2AR.

  • Inosine dose-dependently induces A2AR-mediated ERK1/2 phosphorylation.

  • Adenosine and inosine differ in their signaling biases at the A2AR.

Abstract

Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10 s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15 h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A2A receptor (A2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A2AR and subsequent activation of downstream signaling events. Inosine-mediated A2AR activation leads to cAMP production with an EC50 of 300.7 μM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38 μM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A2AR activation in vivo.

Introduction

Inosine is an endogenous purine nucleoside formed by deamination of adenosine. As with adenosine, it is produced and released into the extracellular space during normal cell metabolism. Adenosine has a short half-life of less than 10 s in vivo [1] and is rapidly deaminated to inosine by adenosine deaminase. Inosine has a much longer in vivo half-life than adenosine of approximately 15 h [2]. In interstitial fluids, the basal level of inosine is in the micromolar range and can be two to seven times that of adenosine [3], [4], [5], [6]. During ischemia and sepsis interstitial inosine levels rise dramatically and in certain disease states can be greater than 1 mM [7] and remain elevated longer than those of adenosine [8]. Consequently, inosine levels become markedly greater than those of adenosine under disease conditions (> 10-fold; [3], [4], [5], [6]). The biological significance of the high levels of inosine in vivo is poorly understood.

As has been observed for adenosine, inosine exerts a wide variety of anti-inflammatory and immunomodulatory effects in vivo and in vitro. These include inhibition of proinflammatory cytokine and chemokine production [9], [10], [11], stimulation of anti-inflammatory cytokine production [9], protection from TNBS-induced colitis [12], LPS-induced endotoxemia [13], [14], LPS-induced acute lung injury [10], glycodeoxycholic acid-induced acute pancreatitis [15], streptozotocin-induced and non-obese type 1 diabetes [16] as well as improvement of islet transplant survival [17]. However, the molecular mechanisms underlying the anti-inflammatory and immunomodulatory effects of inosine are incompletely understood. They do not require cellular uptake of inosine, suggesting involvement of cell surface receptors in this process. However, the presence of cell-surface receptors specific for inosine has not been documented. It is widely stated that the biological effects of inosine are mediated through ligation of specific membrane-bound G protein-coupled receptors (GPCRs) termed P1-purinoceptors, also known as adenosine receptors (AR; [18]).

There are four pharmacologically distinct adenosine receptor subtypes, termed A1R, A2AR, A2BR and A3R [19]. A2AR and A2BR are coupled to the stimulatory G protein Gαs while A1R and A3R are coupled to inhibitory G protein Gi [19]. Thus, adenosine occupation at the A2AR and A2BR leads to an increase in intracellular cAMP levels, whereas adenosine ligation at the A1R and A3R results in a reduction in intracellular cAMP levels. Among the four AR subtypes, A2AR is the most effective in downregulating inflammation through modulation of intracellular cAMP levels. Although inosine is a functional agonist for A1R and A3R, with EC50 values of 290 μM and 0.25 μM, respectively, inosine-mediated anti-inflammatory effects resemble those of the activated A2AR. In vivo studies utilizing receptor knockout mice demonstrate that the anti-inflammatory properties of inosine are mediated in part through the A2AR [20], [21]. Additionally, inosine-mediated inhibition of the production of the pro-inflammatory cytokine TNF-α following LPS-stimulation of peritoneal macrophages is partially reversed by the A2AR-selective antagonist DMPX in vitro [9]. Nevertheless, efforts directed toward a comprehensive demonstration of inosine as a functional agonist at the A2AR at the molecular and cellular level or inosine's ability to bind directly to the A2AR have been unsuccessful. Hence, inosine is largely considered to be inactive at the A2AR [22] in spite of a large body of in vivo data suggesting otherwise.

To understand the molecular mechanisms underlying the inosine-mediated anti-inflammatory and immunomodulatory effects, we examined inosine agonism at both the human and mouse A2ARs utilizing a variety of cell-based and membrane-based assays. Here we demonstrate that inosine not only binds to the A2AR but also activates A2AR-mediated cAMP production and ERK1/2 phosphorylation. To the best of our knowledge, this is the first comprehensive demonstration of inosine functional agonism at the A2AR. Our data suggest the existence of a sustained A2AR receptor signaling mechanism that is driven by inosine long after its more potent predecessor, adenosine, has been degraded, adding a new dimension to the anti-inflammatory and immunomodulatory role of the A2AR.

Section snippets

Materials

NECA, CGS 21680, and ZM 241385 were purchased from Tocris Biosciences. [3H]CGS 21680 was purchased from PerkinElmer Corporation. Rolipram, adenosine, inosine, inosine monophosphate, xanthine, hypoxanthine, adenosine 5′ [α,β-methylene] diphosphate and dipyridamole were purchased from Sigma-Aldrich.

Cell culture

CHO-K1 cells stably expressing human A2AR (CHO-hA2AR; [23]) and human A2BR (CHO-hA2BR; [23]) were grown in DMEM/F-12 (1:1) supplemented with 10% FBS, 2 mM glutamine and G418 (0.2 mg/ml). CHO-K1 cells

Inosine generates an A2AR-selective, inverse agonist-sensitive dynamic mass redistribution (DMR) signal in CHO-hA2AR cells

Signaling through the GPCR family of receptors is complex. Receptor coupling to more than one G protein subunit leading to different signaling outcomes is common. The A2AR couples to two different classes of Gα subunits. A2AR coupling to the Gαs and Gαolf subunits that belong to the Gs class stimulates adenylate cyclase to increase intracellular cAMP upon agonist engagement at the receptor [23]. Agonist engagement at the A2AR also leads to ERK1/2 phosphorylation via either cAMP dependent or

Discussion

Inosine is a ubiquitous purine nucleoside that exerts anti-inflammatory and immunomodulatory effects. Inosine and metabolically stable analogs of inosine [31], [32] utilize adenosine receptors to modulate anti-inflammatory effects. There are four adenosine receptor (AR) subtypes termed A1R, A2AR, A2BR and A3R. Among them, A2AR is the most effective receptor in downregulating inflammation [33], [34]. The role of the A2AR, however, is less clear in the context of inosine-mediated

Conflict of interest

The authors declare that they have no conflict of interest. Research reported in this publication was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R21AI105518. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Acknowledgments

We gratefully acknowledge Dr. Karl-Norbert Klotz for providing CHO-hA2AR and CHO-hA2BR cells and Dr. Jan. Rydzewski for HPLC analysis. This work was supported in part by NIH grant 5R21AI105518 from the National Institute of Allergy and Infectious Diseases.

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