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    Definition

    Adenosine is a signalling molecule which, by activating specific membrane receptors, acts as an important player during brain insults such as ischemia. or demyelinating injuries. Here we review data in the literature describing A2B receptor-mediated effects in preclinical in vitro and in vivo models of cerebral ischemia and myelination that point to A2B receptor ligands as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.

    1. Introduction

    Adenosine acts through the activation of four different purinergic P1 receptors: A1, A2A, A2B, and A3 adenosine receptors (A1Rs, A2ARs, A2BRs, and A3Rs, respectively), all belonging to the G-protein coupled, metabotropic receptor family [1].

    The most widely recognized adenosine signaling is through the activation of A1Rs, which inhibits adenylyl cyclase (AC) through Gi/o protein activation [2]. A1Rs are dominant in the central nervous system (CNS), where they inhibit neurotransmission and mediate sedative, anticonvulsant, anxiolytic, and locomotor depressant effects [3].

    The A2AR subtype is known to stimulate AC [2] being coupled to Gs proteins [1]. At central level, the functional effect of A2AR activation is at variance from A1Rs, as they are reported to enhance glutamate release [4][5].  In the periphery, A2ARs are highly expressed in inflammatory cells including lymphocytes, granulocytes, and monocytes/macrophages, where their activation reduces pro-inflammatory cytokine production, i.e., tumor necrosis factor-alpha (TNFα), interleukin-1β (IL-1 β), and IL-6 [6] and enhances the release of anti-inflammatory mediators, such as IL-10 [7].

    The relatively new A3R subtype is coupled to Gi/o proteins and inhibits AC but, under particular conditions or in different cell types, activation of Gq/11 by A3R agonists has also been reported [1]. Most of the cell types of the immune system express functional A3Rs on their surface [8] and its activation is one of the most powerful stimuli for mast cell degranulation.

    2. A2B Adenosine Receptors (A2BRs)

    This adenosine receptor subtype is somewhat the most enigmatic and less studied among the four P1 receptors. Although it was cloned in 1995 [9], a pharmacological and physiological characterization of A2BRs has long been precluded by the lack of suitable ligands able to discriminate among the other adenosine receptor subtypes [10].

    The distribution of A2BRs in the CNS on neurons and glia is scarce but widespread, whereas in the periphery, abundant expression of A2BRs is observed in the bronchial epithelium, vascular beds, smooth muscles, mast cells, monocytes, and digestive tracts such as ileum and colon [1]. The activation of A2BRs stimulates Gs and, in some cases, Gq/11 proteins, thus enhancing intracellular [cAMP] or [IP3], respectively [1]. As mentioned above for the cognate A2AR subtype, in addition to brain cells and endothelial cells, A2BRs are present on hematic cells, such as lymphocytes and neutrophils, with the highest expression levels on macrophages [11]. Here, A2B receptors in most cases are coexpressed with A2ARs and their activation exerts anti-inflammatory effects, inhibiting vascular adhesion and migration of inflammatory cells [12].

    Differently from the high affinity A1Rs, A2ARs and A3Rs, which are activated by physiological levels of extracellular adenosine (low nM and high nM, respectively [13]), the A2BR needs much higher adenosine concentrations (in the µM range) reached only in conditions of tissue damage or injury. Such a low affinity of A2BRs for the endogenous agonist implies that they represent a good therapeutic target, since they are activated only by high adenosine efflux reached under pathological conditions or injury, when a massive release of adenosine occurs [14].

    3. A2BRs and Oligodendrogliogenesis

    We recently and originally demonstrated that A2BRs are crucially involved in oligodendrocyte progenitor cell (OPC) maturation. We found that the selective A2BR agonists BAY60-6582 (10 μM) and P453 (500 nM) inhibited the differentiation of purified primary OPC cultures, as demonstrated by the reduced expression of myelin basic protein (MBP) and myelin associated glycoprotein (MAG). We also demonstrated that A2BR activation reversibly inhibits tetraethylammonium- (TEA-) sensitive, sustained IK, and 4-amynopyridine- (4-AP) sensitive, transient IA, conductances [15]. As IK are known to be necessary to OPC maturation [16], this could be one of the mechanisms by which A2BRs inhibit myelin production. These results are similar to what was observed in cultured OPCs exposed to the A2AR agonist CGS21680, as demonstrated by us in a previous work [17][18].

    4. A2BRs and brain ischemia

    Brain ischemia results from a permanent or transient reduction in cerebral blood flow mostly due to the occlusion of a brain artery. The consequent reduction of blood and/or oxygen supply to the brain leads to neuronal death caused by excessive glutamate release [19]. This early excitotoxic damage is followed by a secondary chronic phase of neuroinflammation that develops hours and days after ischemia. During stroke, adenosine is released in massive amounts [13][20]. The block of A2BRs is neuroprotective as it counteracts glutamate overload by preserving the inhibitory role of A1Rs on neurotransmission [21][22][23], as demonstrated by us in an in vitro model of brain ischemia reproduced in rat hippocampal slices by oxygen and glucose deprivation (OGD)[22]. The selective A2BR antagonists PSB-603 (50 nM) and by MRS1754 (200 nM) prevents irreversible synaptic failure and anoxic depolarization (AD) appearance produced by a severe, 7 min, OGD event in CA1 hippocampal slices [22].

    However, beyond neuroprotection exerted by A2BR antagonists acting at the neuro-glial level, evidence in the literature points to a beneficial role exerted by A2BR agonists acting on the same receptor subtype expressed on blood vessels and inflammatory cells [11][24]. Indeed, post-treatment with intravenous BAY60-6583 (1 mg/kg) reduces lesion volume and attenuates brain swelling and blood–brain barrier disruption at 24 h after ischemia induced by transient (2 h) middle cerebral artery occlusion (tMCAo) [25]. Additionally, in the same work, BAY60-6583 mitigates sensorimotor deficits in the presence of tPA and inhibits tPA-enhanced matrix metalloprotease-9 activation, thus decreasing BBB permeability 24 h after ischemia [25].

    Our group of research contributed to the field by demonstrating that the chronic treatment with BAY60-6583, administered intraperitoneally twice/day for 7 days at the dose of 0.1 mg/kg, from 4 h after focal ischemia induced by tMCAo, since one day after ischemia protects from neurological deficit. Seven days after ischemia it protects from ischemic brain damage, neuronal death, microglia activation, and astrocyte alteration [26]. Interestingly, in the same paper, it was demonstrated that, 7 days after ischemia, the A2B agonist decreases TNF-α and increases IL-10 levels in the blood. 

    5. A2BRs and demyelinating diseases

    Demyelination occurs in a variety of pathological conditions affecting central or peripheral nervous systems. As an example, myelin disorganization in caudate/putamen striatal nuclei have been reported by us [27] and others [28][29]. Furthermore, chronic demyelinating diseases, such as multiple sclerosis (MS), are highly invalidating pathologies with elevated incidence among the “under 40” population worldwide [30], but an efficacious therapy is still lacking.

    crucial role of adenosine, and in particular of A2AR and/or A2BR subtypes, in demyelinating pathologies have been postulated.

    Under these conditions, excessive signaling by excitatory neurotransmitters like glutamate may be deleterious to neurons and oligodendroglia by exacerbating excitotoxicity and contributing to brain injury. For this reason, the inhibitory effect on glutamate release described above for antagonists at both A2R subtypes could prove protective. This was indeed the case, as demonstrated by Chen and colleagues [31] and by Wei and co-workers [32] who reported that A2AR and A2BR antagonists, respectively, alleviated the clinical symptoms of EAE and prevented demyelination and CNS damage. Recent data by Liu and co-workers [33] confirmed that A2BR activation seems to participate in EAE-induced damage as BAY60-6583 reverted the protective effects, i.e., reduced inflammatory cell infiltration and demyelination, exerted by mesenchymal stem cell therapy in EAE mice. Of note, the above results demonstrating a deleterious role of A2BRs in demyelinating diseases are in agreement with our in vitro data demonstrating that A2BR blockade [15], as well as A2AR antagonism [17], facilitates OPC differentiation in vitro.

    However, things are probably more complicated as suggested by the fact that, again, A2R-mediated actions are mainly anti-inflammatory when observed in a longer time-span. Indeed, genetically modified A2AR-/- EAE mice are more prone to EAE-induced damage [34], and the A2AR agonist CGS61680 ameliorates EAE by reducing Th1 lymphocyte activation and cytokine-induced BBB dysfunction [35].

    6. Conclusions

    In conclusion, results underlie that after hypoxia/ischemia, brain injury results from a complex sequence of pathophysiological events that evolve over time—a primary acute mechanism of excitotoxicity and periinfarct depolarizations followed by a secondary brain injury activation triggered by protracted neuroinflammation. Information so far acquired indicates that adenosine A2BRs located on any cell type of the brain and on vascular and blood cells partake in either salvage or demise of the tissue after a stroke, including protracted demyelination.

    Thus, they all represent important targets for drugs having different therapeutic time-windows after stroke. The final protective outcome for an agonist versus antagonist compound depends on time of administration and district of activation of the receptor targeted by the drug.

    This entry is adapted from 10.3390/ijms21249697

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