Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.

Acetylcholine modulates K+ and Na+ currents in human basal forebrain cholinergic neuroblasts through an autocrine/paracrine mechanism.

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced IK and reduced INa currents by stimulating Gi -coupled M2 or phospholipase C-coupled M3 receptors, respectively. Inhibition of acetylcholine esterase activity by neostigmine unveiled a spontaneous acetylcholine release from hfNBM neuroblasts that might account for an autocrine/paracrine signaling during human brain development. Present data provide the first description of cholinergic effects in human NBM neurons and point to a role of acetylcholine as an autocrine/paracrine modulator of voltage-dependent channels. Our research could be of relevance in understanding the mechanisms of cholinergic system development and functions in the human brain, either in health or disease.

Protective effects of carbonic anhydrase inhibition in brain ischaemia in vitro and in vivo models.

Ischaemic stroke is a leading cause of death and disability. One of the major pathogenic mechanisms after ischaemia includes the switch to the glycolytic pathway, leading to tissue acidification. Carbonic anhydrase (CA) contributes to pH regulation. A new generation of CA inhibitors, AN11-740 and AN6-277 and the reference compound acetazolamide (ACTZ) were investigated in two models of brain ischaemia: in rat hippocampal acute slices exposed to severe oxygen, glucose deprivation (OGD) and in an in vivo model of focal cerebral ischaemia induced by permanent occlusion of the middle cerebral artery (pMCAo) in the rat. In vitro, the application of selective CAIs significantly delayed the appearance of anoxic depolarisation induced by OGD. In vivo, sub-chronic systemic treatment with AN11-740 and ACTZ significantly reduced the neurological deficit and decreased the infarct volume after pMCAo. CAIs counteracted neuronal loss, reduced microglia activation and partially counteracted astrocytes degeneration inducing protection from functional and tissue damage.

Uncovering the Mechanisms of Adenosine Receptor-Mediated Pain Control: Focus on the A3 Receptor Subtype.

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

Role of Carbonic Anhydrase in Cerebral Ischemia and Carbonic Anhydrase Inhibitors as Putative Protective Agents.

Ischemic stroke is a leading cause of death and disability worldwide. The only pharmacological treatment available to date for cerebral ischemia is tissue plasminogen activator (t-PA) and the search for successful therapeutic strategies still remains a major challenge. The loss of cerebral blood flow leads to reduced oxygen and glucose supply and a subsequent switch to the glycolytic pathway, which leads to tissue acidification. Carbonic anhydrase (CA, EC 4.2.1.1) is the enzyme responsible for converting carbon dioxide into a protons and bicarbonate, thus contributing to pH regulation and metabolism, with many CA isoforms present in the brain. Recently, numerous studies have shed light on several classes of carbonic anhydrase inhibitor (CAI) as possible new pharmacological agents for the management of brain ischemia. In the present review we summarized pharmacological, preclinical and clinical findings regarding the role of CAIs in strokes and we discuss their potential protective mechanisms.

A2B Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination.

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing A2BR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, A2BRs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by A2BRs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the A2BRs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.

Equilibrative nucleoside transporter ENT1 as a biomarker of Huntington disease.

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.

Time-course of protection by the selective A2A receptor antagonist SCH58261 after transient focal cerebral ischemia.

Evidence indicates that the adenosine A2A receptor subtype is of critical importance in stroke. In previous studies, in the model of permanent middle cerebral artery occlusion (pMCAo), the adenosine A2A receptor antagonist, SCH58261, administered soon after ischemia, proved protective against excessive glutamate outflow in the first 4 h after ischemia and against neurological deficit and tissue damage evaluated 24 h after pMCAo. In the present work, we investigated if neuroprotective effect of SCH58261 was maintained 7 days after transient MCAo (tMCAo). SCH58261 (0.01 mg/kg, i.p.), administered twice/day for 7 days, protected from neurological deficit 1 day after tMCAo, but no more after 5 and 7 days. Two days after tMCAo, SCH58261 did not reduce blood cell infiltration, evaluated as HIS-48 positive cells, into ischemic striatal and cortical tissue. Moreover, 7 days after tMCAo, SCH58261 has not protected ischemic areas from damage and has not ameliorated myelin organization into the ischemic striatum. Protection by the A2A receptor antagonist 24 h after ischemia is attributable to reduced excitotoxicity. Seven days after ischemia the early protective effect of the A2A receptor antagonist likely has been overwhelmed by a secondary damage due to blood cell infiltration and neuroinflammation.

Adenosine A2A receptors modulate acute injury and neuroinflammation in brain ischemia.

The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A(2A) receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A(2A) receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A(2A) receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A(2A) receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A(2A) receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A(2A) receptor agonists a wide therapeutic time-window of hours and even days after stroke.

UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors.

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K(+) currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G-protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K(+) currents. Specifically, receptor stimulation by its agonist UDP-glucose enhances delayed rectifier K(+) currents without affecting transient K(+) conductances. This effect was observed in a subpopulation of OPCs and immature pre-OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP-glucose on K(+) currents is concentration-dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K(+) channel blocker tetraethyl-ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K(+) currents is responsible for GPR17-induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post-traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.

Pharmacological Characterization of P626, a Novel Dual Adenosine A2A/A2B Receptor Antagonist, on Synaptic Plasticity and during an Ischemic-like Insult in CA1 Rat Hippocampus.

In recent years, the use of multi-target compounds has become an increasingly pursued strategy to treat complex pathologies, including cerebral ischemia. Adenosine and its receptors (A1AR, A2AAR, A2BAR, A3AR) are known to play a crucial role in synaptic transmission either in normoxic or ischemic-like conditions. Previous data demonstrate that the selective antagonism of A2AAR or A2BAR delays anoxic depolarization (AD) appearance, an unequivocal sign of neuronal injury induced by a severe oxygen-glucose deprivation (OGD) insult in the hippocampus. Furthermore, the stimulation of A2AARs or A2BARs by respective selective agonists, CGS21680 and BAY60-6583, increases pre-synaptic neurotransmitter release, as shown by the decrease in paired-pulse facilitation (PPF) at Schaffer collateral-CA1 synapses. In the present research, we investigated the effect/s of the newly synthesized dual A2AAR/A2BAR antagonist, P626, in preventing A2AAR- and/or A2BAR-mediated effects by extracellular recordings of synaptic potentials in the CA1 rat hippocampal slices. We demonstrated that P626 prevented PPF reduction induced by CGS21680 or BAY60-6583 and delayed, in a concentration-dependent manner, AD appearance during a severe OGD. In conclusion, P626 may represent a putative neuroprotective compound for stroke treatment with the possible translational advantage of reducing side effects and bypassing differences in pharmacokinetics due to combined treatment.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke.

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre's behavioral evaluations before project's interventional phase.

P2 receptor antagonists prevent synaptic failure and extracellular signal-regulated kinase 1/2 activation induced by oxygen and glucose deprivation in rat CA1 hippocampus in vitro.

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen-activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS, unselective, 30 µm), N( 6) -methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, selective for P2Y(1) receptor, 10 µm), Brilliant Blue G (BBG, selective for P2X(7) receptor, 1 µm), and 5-[[[(3-phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid (A-317491, selective for P2X(3) receptor, 10 µm), and of the newly synthesized P2X(3) receptor antagonists 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)adenine (PX21, 1 µm) and 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N( 6)-methyladenine (PX24, 1 µm), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 µm), MRS2179 (10 µm), and BBG (1 µm). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal-regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A-317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126, 10 µm) and α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile (SL327, 10 µm) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2.

Selective adenosine A2A receptor agonists and antagonists protect against spinal cord injury through peripheral and central effects.

BACKGROUND: Permanent functional deficits following spinal cord injury (SCI) arise both from mechanical injury and from secondary tissue reactions involving inflammation. Enhanced release of adenosine and glutamate soon after SCI represents a component in the sequelae that may be responsible for resulting functional deficits. The role of adenosine A2A receptor in central ischemia/trauma is still to be elucidated. In our previous studies we have demonstrated that the adenosine A2A receptor-selective agonist CGS21680, systemically administered after SCI, protects from tissue damage, locomotor dysfunction and different inflammatory readouts. In this work we studied the effect of the adenosine A2A receptor antagonist SCH58261, systemically administered after SCI, on the same parameters. We investigated the hypothesis that the main action mechanism of agonists and antagonists is at peripheral or central sites. METHODS: Spinal trauma was induced by extradural compression of SC exposed via a four-level T5-T8 laminectomy in mouse. Three drug-dosing protocols were utilized: a short-term systemic administration by intraperitoneal injection, a chronic administration via osmotic minipump, and direct injection into the spinal cord. RESULTS: SCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI), reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK) 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours), these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the neuroprotective effects of the A2A agonist are due to A2A receptor desensitization. When the A2A antagonist and agonist were centrally injected into injured SC, only SCH58261 appeared neuroprotective, while CGS21680 was ineffective. CONCLUSIONS: Our results indicate that the A2A antagonist protects against SCI by acting on centrally located A2A receptors. It is likely that blockade of A2A receptors reduces excitotoxicity. In contrast, neuroprotection afforded by the A2A agonist may be primarily due to peripheral effects.

A2 B Adenosine Receptors and Sphingosine 1-Phosphate Signaling Cross-Talk in Oligodendrogliogenesis.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain. Impairments in the process of myelination, or demyelinating insults, might cause chronic diseases such as multiple sclerosis (MS). Under physiological conditions, remyelination is an ongoing process throughout adult life consisting in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes (OLs). During pathological events, this process fails due to unfavorable environment. Adenosine and sphingosine kinase/sphingosine 1-phosphate signaling axes (SphK/S1P) play important roles in remyelination processes. Remarkably, fingolimod (FTY720), a sphingosine analog recently approved for MS treatment, plays important roles in OPC maturation. We recently demonstrated that the selective stimulation of A2 B adenosine receptors (A2 B Rs) inhibit OPC differentiation in vitro and reduce voltage-dependent outward K+ currents (I K ) necessary to OPC maturation, whereas specific SphK1 or SphK2 inhibition exerts the opposite effect. During OPC differentiation A2 B R expression increases, this effect being prevented by SphK1/2 blockade. Furthermore, selective silencing of A2 B R in OPC cultures prompts maturation and, intriguingly, enhances the expression of S1P lyase, the enzyme responsible for irreversible S1P catabolism. Finally, the existence of an interplay between SphK1/S1P pathway and A2 B Rs in OPCs was confirmed since acute stimulation of A2 B Rs activates SphK1 by increasing its phosphorylation. Here the role of A2 B R and SphK/S1P signaling during oligodendrogenesis is reviewed in detail, with the purpose to shed new light on the interaction between A2 B Rs and S1P signaling, as eventual innovative targets for the treatment of demyelinating disorders.

Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia.

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischaemia. During ischaemia, adenosine is in a concentration range (muM) that stimulates all four adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)). In recent years, evidence has indicated that the A(2A) receptor subtype is of critical importance in stroke. We have previously shown that 24 h after medial cerebral artery occlusion (MCAo), A(2A) receptors up-regulate on neurons and microglia of ischaemic striatum and cortex and that subchronically administered adenosine A(2A) receptor antagonists protect against brain damage and neurological deficit and reduce activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells. The mechanisms by which A(2A) receptors are noxious during ischaemia still remain elusive. The objective of the present study was to investigate whether the adenosine A(2A) antagonist SCH58261 affects JNK and MEK1/ERK MAPK activation. A further aim was to investigate cell types expressing activated JNK and MEK1/ERK MAPK after ischaemia. We hereby report that the selective adenosine A(2A) receptor antagonist, SCH58261, administered subchronically (0.01 mg/kg i.p) 5 min, 6 and 20 h after MCAo in male Wistar rats, reduced JNK MAPK activation (immunoblot analysis: phospho-JNK54 isoform by 81% and phospho-JNK46 isoform by 60%) in the ischaemic striatum. Twenty-four hours after MCAo, the Olig2 transcription factor of oligodendroglial progenitor cells and mature oligodendrocytes was highly expressed in cell bodies in the ischaemic striatum. Immunofluorescence staining showed that JNK MAPK is maximally expressed in Olig2-stained oligodendrocytes and in a few NeuN stained neurons. Striatal cell fractioning into nuclear and extra-nuclear fractions demonstrated the presence of Olig2 transcription factor and JNK MAPK in both fractions. The A(2A) antagonist reduced striatal Olig 2 transcription factor (immunoblot analysis: by 55%) and prevented myelin disorganization, assessed by myelin-associated glycoprotein staining. Twenty-four hours after MCAo, ERK1/2 MAPK was highly activated in the ischaemic striatum, mostly in microglia, while it was reduced in the ischaemic cortex. The A(2A) antagonist did not affect activation of the ERK1/2 pathway. The efficacy of A(2A) receptor antagonism in reducing activation of JNK MAPK in oligodendrocytes suggests a mechanism of protection consisting of scarring oligodendrocyte inhibitory molecules that can hinder myelin reconstitution and neuron functionality.

Protective Effect of Adenosine A2B Receptor Agonist, BAY60-6583, Against Transient Focal Brain Ischemia in Rat.

Cerebral ischemia is a multifactorial pathology characterized first by an acute injury, due to excitotoxicity, followed by a secondary brain injury that develops hours to days after ischemia. During ischemia, adenosine acts as an endogenous neuroprotectant. Few studies have investigated the role of A2B receptor in brain ischemia because of the low potency of adenosine for it and the few selective ligands developed so far. A2B receptors are scarcely but widely distributed in the brain on neurons, glial and endothelial cells and on hematopoietic cells, lymphocytes and neutrophils, where they exert mainly anti-inflammatory effects, inhibiting vascular adhesion and inflammatory cells migration. Aim of this work was to verify whether chronic administration of the A2B agonist, BAY60-6583 (0.1 mg/kg i.p., twice/day), starting 4 h after focal ischemia induced by transient (1 h) Middle Cerebral Artery occlusion (tMCAo) in the rat, was protective after the ischemic insult. BAY60-6583 improved the neurological deficit up to 7 days after tMCAo. Seven days after ischemia BAY60-6583 reduced significantly the ischemic brain damage in cortex and striatum, counteracted ischemia-induced neuronal death, reduced microglia activation and astrocytes alteration. Moreover, it decreased the expression of TNF-α and increased that of IL-10 in peripheral plasma. Two days after ischemia BAY60-6583 reduced blood cell infiltration in the ischemic cortex. The present study indicates that A2B receptors stimulation can attenuate the neuroinflammation that develops after ischemia, suggesting that A2B receptors may represent a new interesting pharmacological target to protect from degeneration after brain ischemia.

Adenosine A2B receptors inhibit K+ currents and cell differentiation in cultured oligodendrocyte precursor cells and modulate sphingosine-1-phosphate signaling pathway.

Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and we recently demonstrated that adenosine A2A receptors inhibit cell maturation by reducing voltage-dependent K+ currents. No data are available to date about the A2B receptor (A2BR) subtype. The bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) are also crucial modulators of OPC development. An interaction between this pathway and the A2BR is reported in peripheral cells. We studied the role of A2BRs in modulating K+ currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signaling. Our data indicate that the A2BR agonist BAY60-6583 and its new analogue P453 inhibit K+ currents in cultured OPC and the effect was prevented by the A2BR antagonist MRS1706, by K+ channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic (7 days) treatment with the same agonist decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, A2BR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, A2BR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. We conclude that A2BRs inhibit K+ currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs.

Acute visceral pain relief mediated by A3AR agonists in rats: involvement of N-type voltage-gated calcium channels.

ABSTRACT: Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently, their activity has been related to the block of N-type voltage-gated Ca2+ channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing postinflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intracolonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 mL 50% EtOH). Visceral hypersensitivity was assessed by measuring the visceromotor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the postinflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in dorsal root ganglia neurons isolated from either control or DNBS-treated rats. The effect on Ca2+ current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.