Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

The role of activated adenosine receptors in degranulation of human LAD2 mast cells

Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A_2A, A_2B, and A₃, but not A₁, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A_2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A_2A, A_2B, and A₃ ARs. but not A₁AR detectably. We conclude that (a) A₁AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A_2A, A_2B, and A₃ ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.     

IL-6, A1 and A2aR: A crosstalk that modulates BDNF and induces neuroprotection

Several diseases are related to retinal ganglion cell death, such as glaucoma, diabetes and other retinopathies. Many studies have attempted to identify factors that could increase neuroprotection after axotomy of these cells. Interleukin-6 has been shown to be able to increase the survival and regeneration of retinal ganglion cells (RGC) in mixed culture as well as in vivo. In this work we show that the trophic effect of IL-6 is mediated by adenosine receptor (A2aR) activation and also by the presence of extracellular BDNF. We also show that there is a complex cross-talk between IL-6, BDNF, the Adenosine A1 and A2a receptors that results in neuroprotection of retinal ganglion cells.     

Adenosine Regulates the Survival of Avian Retinal Neurons and Photoreceptors in Culture

Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were incubated for 3 days and refed with fresh medium, a large proportion of neurons died in the subsequent 3 days of culture. This cell death was prevented by preincubation of cultures for at least 24h with adenosine plus the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine uptake blocker nitrobenzylthioinosine (NBI), the adenosine A_2A receptor agonist 2-[4-(2-carboxyethyl) phenethylamino]-5-N-ethylcarboxamidoadenosine (CGS21680), or the permeant cyclic AMP analog 8-bromo cyclic AMP, but not the A₁ receptor agonist cyclohexyladenosine (CHA). Adenosine deaminase induced cell death when added to culture medium, and this effect was prevented by EHNA. Cell death was not observed when the medium was replaced by a conditioned medium from sister cultures. The data strongly suggest that adenosine regulates the survival of developing retinal neurons by a long-term activation of A_2A receptors and the increase of cyclic AMP levels.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.     

Protection from pulmonary ischemia-reperfusion injury by adenosine A2A receptor activation

Background

Lung ischemia-reperfusion (IR) injury leads to significant morbidity and mortality which remains a major obstacle after lung transplantation. However, the role of various subset(s) of lung cell populations in the pathogenesis of lung IR injury and the mechanisms of cellular protection remain to be elucidated. In the present study, we investigated the effects of adenosine A_2A receptor (A_2AAR) activation on resident lung cells after IR injury using an isolated, buffer-perfused murine lung model.

Methods

To assess the protective effects of A_2AAR activation, three groups of C57BL/6J mice were studied: a sham group (perfused for 2 hr with no ischemia), an IR group (1 hr ischemia + 1 hr reperfusion) and an IR+ATL313 group where ATL313, a specific A_2AAR agonist, was included in the reperfusion buffer after ischemia. Lung injury parameters and pulmonary function studies were also performed after IR injury in A_2AAR knockout mice, with or without ATL313 pretreatment. Lung function was assessed using a buffer-perfused isolated lung system. Lung injury was measured by assessing lung edema, vascular permeability, cytokine/chemokine activation and myeloperoxidase levels in the bronchoalveolar fluid.

Results

After IR, lungs from C57BL/6J wild-type mice displayed significant dysfunction (increased airway resistance, pulmonary artery pressure and decreased pulmonary compliance) and significant injury (increased vascular permeability and edema). Lung injury and dysfunction after IR were significantly attenuated by ATL313 treatment. Significant induction of TNF-α, KC (CXCL1), MIP-2 (CXCL2) and RANTES (CCL5) occurred after IR which was also attenuated by ATL313 treatment. Lungs from A_2AAR knockout mice also displayed significant dysfunction, injury and cytokine/chemokine production after IR, but ATL313 had no effect in these mice.

Conclusion

Specific activation of A_2AARs provides potent protection against lung IR injury via attenuation of inflammation. This protection occurs in the absence of circulating blood thereby indicating a protective role of A_2AAR activation on resident lung cells such as alveolar macrophages. Specific A_2AAR activation may be a promising therapeutic target for the prevention or treatment of pulmonary graft dysfunction in transplant patients.     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.     

Modulatory Role of Adenosine Receptors in Insect Motor Nerve Terminals

The effects of adenosine and ATP were studied on blowfly larvae Calliphora vicina neuromuscular preparation. Adenosine diminished (IC₅₀ = 40 ± 3 M) the amplitude of nerve-evoked postsynaptic currents (EPSCs) and slightly decreased the frequency of spontaneous currents without affecting their amplitude. EPSCs were slightly reduced by ATP, and this effect was prevented by concanavalin A. Presynaptic inhibition by adenosine was temperature-dependent and insensitive to pertussis toxin. A₁ agonists of vertebrate adenosine receptor CPA and NECA failed to reproduce the effect of adenosine, and 2-CADO enhanced the EPSCs. A₁ antagonist DPCPX competitively inhibited adenosine action. A₂ agonist DPMA potentiated EPSCs, and its effect was abolished by A₂ antagonist DMPX. Adenosine and ATP failed to affect the nonquantal release of glutamate. The results show for the first time the presence of presynaptic adenosine receptors regulating transmitter release at insect motor nerve terminals and point to differences in pharmacological properties of adenosine receptor subtypes in insects and vertebrates.     

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport

A₁ adenosine receptors (ARs) reduce, and A₂ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A₁AR and A₂AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A₁, A_2A and A₃AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A₁ARs, CGS 21680 and DPMA for A_2AARs, and Cl-IB-MECA and IB-MECA for A₃ARs. Stimulation of A₁, A_2A and A₃ARs elevated Ca²⁺, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A₁, A_2A and A₃AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A₁, A_2A and A₃ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca²⁺ and volume, but likely by actions on separate cell targets. Reprint requests should be addressed to: Dr. Mortimer M. Civan, Dept. of Physiology, University of Pennsylvania, Richards Building, Philadelphia, PA 19104-6085. [Tel.: (215)-898-8773; Fax: (215)-573-5851]     

Anti-inflammatory effects of purine nucleosides, adenosine and inosine, in a mouse model of pleurisy: evidence for the role of adenosine A2 receptors

Adenosine and its metabolite, inosine, have been described as molecules that participate in regulation of inflammatory response. The aim of this study was to investigate the effect of adenosine and inosine in a mouse model of carrageenan-induced pleurisy as well as the participation of adenosine receptors in this response. Injection of carrageenan into the pleural cavity induced an acute inflammatory response characterized by leukocyte migration, pleural exudation, and increased release of interleukin-1β and tumor necrosis factor-α in pleural exudates. The treatment with adenosine (0.3–100 mg/kg, i.p.) and inosine (0.1–300 mg/kg, i.p.) 30 min before carrageenan injection reduced significantly all these parameters analyzed. Our results also demonstrated that A_2A and A_2B receptors seem to mediate the adenosine and inosine effects observed, since pretreatment with selective antagonists of adenosine A_2A (ZM241385) and A_2B (alloxazine) receptors, reverted the inhibitory effects of adenosine and inosine in pleural inflammation. The involvement of A₂ receptors was reinforced with adenosine receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment, since its anti-inflammatory effects were reversed completely and partially with ZM241385 and alloxazine injection, respectively. Moreover, the combined treatment with subeffective dose of adenosine (0.3 mg/kg) and inosine (1.0 mg/kg) induced a synergistic anti-inflammatory effect. Thus, based on these findings, we propose that inosine contributes with adenosine to exert anti-inflammatory effects in pleural inflammation, reinforcing the notion that endogenous nucleosides play an important role in controlling inflammatory diseases. This effect is likely mediated by the activation of adenosine A₂ subtype receptors and inhibition of production or release of pro-inflammatory cytokines.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

Background

The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives

The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results

Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A_2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A_2A is the adenosine receptor present in platelets; it is known that inosine is not an A_2A ligand. Docking of adenosine and inosine inside A_2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH₂ of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A_2A receptor.

Conclusion

Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.     

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

The A₃ adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A₃AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A₃AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A₃AR antagonist for all three species tested, albeit a little less selective for mouse and rat A₃AR in comparison to the human A₃AR. DPTN’s K_i values at respective A₁, A_2A, A_2B, and A₃ receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A₃ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A₃AR antagonists in rats and mice, we also re-examined other commonly used and selective A₃AR antagonists under the same experimental conditions. The K_i values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A₃ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [¹²⁵I]I-AB-MECA binding to mouse and rat A₃ARs, while potent human A₃AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A₃AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A₃AR-related mechanism or function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5.     

Adenosine A2A Receptor Up-Regulates Retinal Wave Frequency via Starburst Amacrine Cells in the Developing Rat Retina

Background

Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine''s regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A_2A receptor (A_2AR) regulates retinal waves and whether A_2AR regulation of retinal waves acts via presynaptic SACs.

Methodology/Principal Findings

We showed that A_2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A_2AR decreased the frequency of spontaneous Ca²⁺ transients, suggesting that endogenous A_2AR may up-regulate wave frequency. To investigate whether A_2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca²⁺ transient frequency was increased by expressing wild-type A_2AR (A_2AR-WT) in SACs, suggesting that A_2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A_2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC''s excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A_2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A_2AR mutant (A_2AR-ΔC) in SACs, the wave frequency was reduced compared to the A_2AR-WT, but was similar to the control, suggesting that the full-length A_2AR in SACs is required for A_2AR up-regulation of retinal waves.

Conclusions/Significance

A_2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by coupling with the downstream intracellular signaling, A_2AR may have a great capacity to modulate patterned spontaneous activity during neural circuit refinement.     

Pharmacology of the Adenosine A3 Receptor in the Vasculature and Essential Hypertension

Background

Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A₃ receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A₃ receptor agonists in coronary blood vessels using the isolated perfused heart preparation.

Methods

mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A₃ receptor mediated coronary vasodilation in the rat heart.

Results

Adenosine A₃ receptor agonists induced coronary vasodilation. The expression of adenosine A₃ receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A₃ receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A₃ receptor agonists.

Conclusions

This study demonstrated alterations in the expression of adenosine A₃ receptors occurred in a tissue specific mode, and reduced adenosine A₃ receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A₃ receptor in hypertensive hearts suggest that adenosine A₃ receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A₃ receptor agonists.     

Adenosine activates thermogenic adipocytes

Brown or beige fat activation can cause potent anti-obesity and anti-diabetic effects. In a study recently published in Nature, Gnad et al. show that adenosine is a novel activator of brown and beige fat that acts through the A_2A receptor.Brown fat is a thermogenic type of adipose tissue containing abundant mitochondria and multilocular lipid droplets, and is uniquely suited and required for cold tolerance and body weight control¹. Upon brown fat activation, intracellular cyclic AMP (cAMP) levels rise rapidly, leading to lipolysis and upregulation of uncoupling protein 1 (UCP1) which initiates uncoupling of mitochondrial respiration and heat generation^2,3. Additionally, beige or brite fat is a form of white fat that can be “browned” and induced to engage in respiratory uncoupling similarly as brown fat. Activated brown and beige fat consume substantial amounts of metabolic substrate, leading to powerful anti-obesity and anti-diabetic effects in mice. Adult humans possess brown and beige fat, making these tissues potentially important targets for treating obesity and metabolic diseases⁴. Activation of brown and beige fat occurs through several mechanisms including cold, adrenergic signaling, and hormone signaling⁴. However, a critical question remains of which pathway is altered therapeutically, as targeting these known activation pathways may cause discomfort, have undesirable side effects, or be insufficient alone to exert anti-obesity effects in humans. Thus, the need to discover novel pathways for brown and beige fat thermogenic activation persists.Adenosine is a purine nucleoside that alters cAMP signaling in several tissues^5,6. There is a ubiquitous presence of adenosine in the extracellular space, and adenosine levels can be increased extracellularly or intracellularly through conversion of adenine nucleotides. Adenosine binds to four P1 G-protein-coupled receptor subtypes, which include the inhibitory receptors A₁ and A₃ and the stimulatory receptors A_2A and A_2B⁷. Inhibitory receptor activation leads to increased adenylate cyclase activity and decreased cAMP, while stimulatory receptor activation has the opposite effect. The distribution of the adenine receptor subtypes varies widely by tissue and species, and the response is dependent on the level of receptor expression, thus the actions of adenosine can be highly disparate depending on the tissue context⁸.In adipose tissue, adenosine was previously found to inhibit cAMP production in studies performed in hamster and rat, where oxygen consumption and lipolysis also decreased^9,10. In a study recently published in Nature, Gnad et al.¹¹ show that adenosine activates lipolysis and the thermogenic program in brown and white human and murine adipocytes (Figure 1). This phenomenon occurs at significantly lower concentrations of adenosine in brown adipocytes than in white adipocytes. The difference is accounted for by differential expression of the A_2A receptor, which is highly expressed in human and murine brown fat relative to white fat. It is also due to the ratio of expression compared to the inhibitory A₁ receptor, which is expressed at low levels in brown fat compared to white fat. Moreover, the authors show that hamster brown fat expresses almost equal amounts of A₁ and A_2A receptors, explaining the lack of activating effects of adenosine in those cells.Open in a separate windowFigure 1Adenosine activates brown or beige adipocytes through the A_2A receptor. Adenosine activates the A_2A receptor, leading to increased cAMP- and PGC-1α-dependent signaling that enhances thermogenesis and lipolysis in brown and beige adipocytes. Crosstalk occurs with adrenergic signaling, which increases cAMP and adenosine levels. Adenosine is released both through efflux from brown adipocytes and through breakdown of extracellular ATP by the ecto-5′-nucleotidase CD73. AR, adrenergic receptor.The determination of the source of the adenosine driving brown fat activation was also pursued. Adenosine could be derived from breakdown of ATP released from sympathetic neurons and brown adipocytes, or from brown adipocyte efflux of adenosine itself. The authors find that activated brown fat both releases adenosine through efflux and converts extracellular ATP to adenosine, as inhibition of the ecto-5′-nucleotidase CD73, which converts ATP to adenosine, only partially blocks the adenosine increase. Noradrenaline also enhances adenosine release from brown fat without any change in extracellular ATP, suggesting an intracellular crosstalk between catecholamine and adenosine signaling.The author further show that A_2A receptor knockout mice exposed to cold exhibit defective thermogenesis, oxygen consumption, and lipolysis, demonstrating the importance of the A_2A receptor in mediating the thermogenic response. Moreover, pharmacological activation with an A_2A receptor agonist increases oxygen consumption in mice, while inhibition with a specific A_2A antagonist decreases cold-induced oxygen consumption. A_2A receptor agonism acts synergistically with noradrenaline in driving lipolysis, revealing an additive thermogenic effect of adenosine with catecholamine signaling. An A_2A agonist protects against diet-induced obesity in mice, leading to increases in lean mass, oxygen consumption, glucose tolerance, and browning of white fat, which signifies the potential therapeutic importance of adenosine signaling. Importantly, overexpression of the A_2A receptor in inguinal white fat increases its browning, indicating that bolstering of A_2A adenosine receptor expression is sufficient to initiate a thermogenic response in the presence of physiological concentration of adenosine.This study reveals an endogenous purinergic signaling pathway that activates brown and beige fat. The critical signaling effector involved is cAMP, therefore the mechanism of activation is similar to those of other agents that act through the sympathetic nervous system or adrenergic signaling. It is unclear whether adenosine would contribute any additional anti-obesity effects than what has already been achieved with these other agents.While this study demonstrates substantial effects of adenosine on thermogenesis, further studies would better elucidate the specificity toward the A_2A receptor in adipocytes. Treatment of A_2A knockout mice with the receptor agonist would help demonstrate whether there are effects of the agonist independent of the A_2A receptor in vivo. Additionally, adipose tissue-specific A_2A knockout mice would reveal whether there are any phenotypic contributions from knockout in other tissues.Another question is whether A_2A receptor agonism could be used chronically as an anti-obesity therapy in the clinic, since adenosine receptors are expressed in many tissues. This study did not explore the potentially confounding effects of A_2A receptor agonism on tissues such as the brain or heart, including complications from vasodilation or cardiotoxicity. Indeed, A_2A receptor antagonists have been used in clinical trials for Parkinson''s disease, highlighting the possible adverse effects of A_2A receptor agonism on the brain. The effect of adenosine on lipolysis also raises the question of whether this pathway is involved in the lipoatrophy caused by nucleoside analogues used as anti-retroviral drugs. Nevertheless, this work adds important information about a cellular signaling pathway that may be leveraged in finding new therapies for obesity and metabolic diseases.     

Effect of Diabetes/Hyperglycemia on the Rat Retinal Adenosinergic System

The early stages of diabetic retinopathy (DR) are characterized by alterations similar to neurodegenerative and inflammatory conditions such as increased neural apoptosis, microglial cell activation and amplified production of pro-inflammatory cytokines. Adenosine regulates several physiological functions by stimulating four subtypes of receptors, A₁AR, A_2AAR, A_2BAR, and A₃AR. Although the adenosinergic signaling system is affected by diabetes in several tissues, it is unknown whether diabetic conditions in the retina can also affect it. Adenosine delivers potent suppressive effects on virtually all cells of the immune system, but its potential role in the context of DR has yet to be studied in full. In this study, we used primary mixed cultures of rat retinal cells exposed to high glucose conditions, to mimic hyperglycemia, and a streptozotocin rat model of type 1 diabetes to determine the effect diabetes/hyperglycemia have on the expression and protein levels of adenosine receptors and of the enzymes adenosine deaminase and adenosine kinase. We found elevated mRNA and protein levels of A₁AR and A_2AAR, in retinal cell cultures under high glucose conditions and a transient increase in the levels of the same receptors in diabetic retinas. Adenosine deaminase and adenosine kinase expression and protein levels showed a significant decrease in diabetic retinas 30 days after diabetes induction. An enzymatic assay performed in retinal cell cultures revealed a marked decrease in the activity of adenosine deaminase under high glucose conditions. We also found an increase in extracellular adenosine levels accompanied by a decrease in intracellular levels when retinal cells were subjected to high glucose conditions. In conclusion, this study shows that several components of the retinal adenosinergic system are affected by diabetes and high glucose conditions, and the modulation observed may uncover a possible mechanism for the alleviation of the inflammatory and excitotoxic conditions observed in diabetic retinas.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Selective Deletion of the A1 Adenosine Receptor Abolishes Heart-Rate Slowing Effects of Intravascular Adenosine In Vivo

Objective

Intravenous adenosine induces temporary bradycardia. This is due to the activation of extracellular adenosine receptors (ARs). While adenosine can signal through any of four ARs (A1AR, A2AAR, A2BAR, A3AR), previous ex vivo studies implicated the A1AR in the heart-rate slowing effects. Here, we used comparative genetic in vivo studies to address the contribution of individual ARs to the heart-rate slowing effects of intravascular adenosine.

Methods and Results

We studied gene-targeted mice for individual ARs to define their in vivo contribution to the heart-rate slowing effects of adenosine. Anesthetized mice were treated with a bolus of intravascular adenosine, followed by measurements of heart-rate and blood pressure via a carotid artery catheter. These studies demonstrated dose-dependent slowing of the heart rate with adenosine treatment in wild-type, A2AAR^−/−, A2BAR^−/−, or A3AR^−/− mice. In contrast, adenosine-dependent slowing of the heart-rate was completely abolished in A1AR^−/− mice. Moreover, pre-treatment with a specific A1AR antagonist (DPCPX) attenuated the heart-rate slowing effects of adenosine in wild-type, A2AAR^−/−, or A2BAR^−/− mice, but did not alter hemodynamic responses of A1AR^−/− mice.

Conclusions

The present studies combine pharmacological and genetic in vivo evidence for a selective role of the A1AR in slowing the heart rate during adenosine bolus injection.