Adenosine A2B receptors differently modulate oligodendrogliogenesis and myelination depending on their cellular localization.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies. Adenosine is emerging as an important player in oligodendrogliogenesis, by activating its metabotropic receptors (A1R, A2AR, A2BR, and A3R). We previously demonstrated that the Gs-coupled A2BR reduced differentiation of primary OPC cultures by inhibiting delayed rectifier (IK) as well as transient (IA) outward K+ currents. To deepen the unclear role of this receptor subtype in neuron-OL interplay and in myelination process, we tested the effects of different A2BR ligands in a dorsal root ganglion neuron (DRGN)/OPC cocultures, a corroborated in vitro myelination assay. The A2BR agonist, BAY60-6583, significantly reduced myelin basic protein levels but simultaneously increased myelination index in DRGN/OPC cocultures analyzed by confocal microscopy. The last effect was prevented by the selective A2BR antagonists, PSB-603 and MRS1706. To clarify this unexpected data, we wondered whether A2BRs could play a functional role on DRGNs. We first demonstrated, by immunocytochemistry, that primary DRGN monoculture expressed A2BRs. Their selective activation by BAY60-6583 enhanced DRGN excitability, as demonstrated by increased action potential firing, decreased rheobase and depolarized resting membrane potential and were prevented by PSB-603. Throughout this A2BR-dependent enhancement of neuronal activity, DRGNs could release factors to facilitate myelination processes. Finally, silencing A2BR in DRGNs alone prevents the increased myelination induced by BAY60-6583 in cocultures. In conclusion, our data suggest a different role of A2BR during oligodendrogliogenesis and myelination, depending on their activation on neurons or oligodendroglial cells.

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons.

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

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.

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.

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.

Ion Channels as New Attractive Targets to Improve Re-Myelination Processes in the Brain.

Multiple sclerosis (MS) is the most demyelinating disease of the central nervous system (CNS) characterized by neuroinflammation. Oligodendrocyte progenitor cells (OPCs) are cycling cells in the developing and adult CNS that, under demyelinating conditions, migrate to the site of lesions and differentiate into mature oligodendrocytes to remyelinate damaged axons. However, this process fails during disease chronicization due to impaired OPC differentiation. Moreover, OPCs are crucial players in neuro-glial communication as they receive synaptic inputs from neurons and express ion channels and neurotransmitter/neuromodulator receptors that control their maturation. Ion channels are recognized as attractive therapeutic targets, and indeed ligand-gated and voltage-gated channels can both be found among the top five pharmaceutical target groups of FDA-approved agents. Their modulation ameliorates some of the symptoms of MS and improves the outcome of related animal models. However, the exact mechanism of action of ion-channel targeting compounds is often still unclear due to the wide expression of these channels on neurons, glia, and infiltrating immune cells. The present review summarizes recent findings in the field to get further insights into physio-pathophysiological processes and possible therapeutic mechanisms of drug actions.

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.

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.

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.

Dexpramipexole Enhances K+ Currents and Inhibits Cell Excitability in the Rat Hippocampus In Vitro.

Dexpramipexole (DEX) has been described as the first-in-class F1Fo ATP synthase activator able to boost mitochondrial bioenergetics and provide neuroprotection in experimental models of ischemic brain injury. Although DEX failed in a phase III trial in patients with amyotrophic lateral sclerosis, it showed favorable safety and tolerability profiles. Recently, DEX emerged as a Nav1.8 Na+ channel and transient outward K+ (IA) conductance blocker, revealing therefore an unexpected, pleiotypic pharmacodynamic profile. In this study, we performed electrophysiological experiments in vitro aimed to better characterize the impact of DEX on voltage-dependent currents and synaptic transmission in the hippocampus. By means of patch-clamp recordings on isolated hippocampal neurons, we found that DEX increases outward K+ currents evoked by a voltage ramp protocol. This effect is prevented by the non-selective voltage-dependent K+ channel (Kv) blocker TEA and by the selective small-conductance Ca2+-activated K+ (SK) channel blocker apamin. In keeping with this, extracellular field recordings from rat hippocampal slices also demonstrated that the compound inhibits synaptic transmission and CA1 neuron excitability. Overall, these data further our understanding on the pharmacodynamics of DEX and disclose an additional mechanism that could underlie its neuroprotective properties. Also, they identify DEX as a lead to develop new modulators of K+ conductances.

Oligodendrocyte precursor cell maturation: role of adenosine receptors.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain and their degeneration leads to demyelinating diseases such as multiple sclerosis. Remyelination requires the differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes but, in chronic neurodegenerative disorders, remyelination fails due to adverse environment. Therefore, a strategy to prompt oligodendrocyte progenitor cell differentiation towards myelinating oligodendrocytes is required. The neuromodulator adenosine, and its receptors (A1, A2A, A2B and A3 receptors: A1R, A2AR, A2BR and A3R), are crucial mediators in remyelination processes. It is known that A1Rs facilitate oligodendrocyte progenitor cell maturation and migration whereas the A3Rs initiates apoptosis in oligodendrocyte progenitor cells. Our group of research contributed to the field by demonstrating that A2AR and A2BR inhibit oligodendrocyte progenitor cell maturation by reducing voltage-dependent K+ currents necessary for cell differentiation. The present review summarizes the possible role of adenosine receptor ligands as potential therapeutic targets in demyelinating pathologies such as multiple sclerosis.

Photobiomodulation of Human Fibroblasts and Keratinocytes with Blue Light: Implications in Wound Healing.

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm2) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.

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.

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.

Experimental Study on Blue Light Interaction with Human Keloid-Derived Fibroblasts.

Keloids are an exuberant response to wound healing, characterized by an exaggerated synthesis of collagen, probably due to the increase of fibroblasts activity and to the reduction of their apoptosis rate: currently no standard treatments or pharmacological therapies are able to prevent keloid recurrence. To reach this goal, in recent years some physical treatments have been proposed, and among them the PhotoBioModulation therapy (PBM). This work analyses the effects of a blue LED light irradiation (410-430 nm, 0.69 W/cm2 power density) on human fibroblasts, isolated from both keloids and perilesional tissues. Different light doses (3.43-6.87-13.7-20.6-30.9 and 41.2 J/cm2) were tested. Biochemical assays and specific staining were used to assess cell metabolism, proliferation and viability. Micro-Raman spectroscopy was used to explore direct effects of the blue LED light on the Cytochrome C (Cyt C) oxidase. We also investigated the effects of the irradiation on ionic membrane currents by patch-clamp recordings. Our results showed that the blue LED light can modulate cell metabolism and proliferation, with a dose-dependent behavior and that these effects persist at least till 48 h after treatment. Furthermore, we demonstrated that the highest fluence value can reduce cell viability 24 h after irradiation in keloid-derived fibroblasts, while the same effect is observed 48 h after treatment in perilesional fibroblasts. Electrophysiological recordings showed that the medium dose (20.6 J/cm2) of blue LED light induces an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol. Overall, these data demonstrate the potentials that PBM shows as an innovative and minimally-invasive approach in the management of hypertrophic scars and keloids, in association with current treatments.

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.

Dexpramipexole blocks Nav1.8 sodium channels and provides analgesia in multiple nociceptive and neuropathic pain models.

Selective targeting of sodium channel subtypes Nav1.7, Nav1.8, and Nav1.9, preferentially expressed by peripheral nociceptors, represents a unique opportunity to develop analgesics devoid of central side effects. Several compounds that target Nav1.7 and Nav1.8 with different degrees of selectivity have been developed and are currently being tested in clinical trials for multiple pain indications. Among these chemicals, benzothiazole-like compounds emerged as potent sodium channel blockers. We evaluated the effects of dexpramipexole, a benzothiazole-bearing drug with pleiotypic neuroactive properties and a good safety profile in humans, on sodium conductances of dorsal root ganglia neurons, as well as in multiple nociceptive and neuropathic pain models. Dexpramipexole blocks TTX-resistant sodium conductances in cultured rat dorsal root ganglion neurons with an IC50 of 294.4 nM, suggesting selectivity towards Nav1.8. In keeping with this, dexpramipexole does not affect sodium currents in dorsal root ganglion neurons from Nav1.8 null mice and acquires binding pose predicted to overlap that of the Nav1.8 channel-selective blocker A-8034637. The drug provides analgesia when parenterally, orally, or topically applied in inflammatory and visceral mouse pain models, as well as in mice affected by neuropathic pain induced by oxaliplatin, nerve constriction, or diabetes. Pain reduction in mice occurs at doses consistent with those adopted in clinical trials. The present findings confirm the relevance of selective targeting of peripheral Nav1.8 channels to pain therapy. In light of the excellent tolerability of dexpramipexole in humans, our results support its translational potential for treatment of pain.

Functional characterization of a novel adenosine A2B receptor agonist on short-term plasticity and synaptic inhibition during oxygen and glucose deprivation in the rat CA1 hippocampus.

Adenosine is an endogenous neuromodulator exerting its biological functions via four receptor subtypes, A1, A2A, A2B, and A3. A2B receptors (A2BRs) are expressed at hippocampal level where they are known to inhibit paired pulse facilitation (PPF), whose reduction reflects an increase in presynaptic glutamate release. The effect of A2BRs on PPF is known to be sensitive not only to A2BR blockade but also to the A1R antagonist DPCPX, indicating that it involves A1R activation. In this study we provide the first functional characterization of the newly synthesized non-nucleoside like A2BR agonist P453, belonging to the amino-3,5-dicyanopyridine series. By extracellular electrophysiological recordings, we demonstrated that P453 mimicked the effect of the prototypical A2BR agonist BAY60-6583 in decreasing PPF at Schaffer collateral-CA1 synapses in rat acute hippocampal slices. This effect was prevented by two different A2BR antagonists, PSB603 and MRS1754, and by the A1R antagonist DPCPX. We also investigated the functional role of A2BR during a 2 min of oxygen and glucose deprivation (OGD) insult, known to produce a reversible fEPSP inhibition due to adenosine A1R activation. We found that P453 and BAY60-6583 significantly delayed the onset of fEPSP reduction induced by OGD and the effect was blocked by PSB603. We conclude that P453 is a functional A2BR agonist whose activation decreases PPF by increasing glutamate release at presynaptic terminals and delays A1R-mediated fEPSP inhibition during a 2-minute OGD insult.

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.