Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

Negative feedback control of neuronal activity by microglia.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

The full activation mechanism of the adenosine A1 receptor revealed by GaMD and Su-GaMD simulations.

The full activation process of G protein-coupled receptor (GPCR) plays an important role in cellular signal transduction. However, it remains challenging to simulate the whole process in which the GPCR is recognized and activated by a ligand and then couples to the G protein on a reasonable simulation timescale. Here, we developed a molecular dynamics (MD) approach named supervised (Su) Gaussian accelerated MD (GaMD) by incorporating a tabu-like supervision algorithm into a standard GaMD simulation. By using this Su-GaMD method, from the active and inactive structure of adenosine A1 receptor (A1R), we successfully revealed the full activation mechanism of A1R, including adenosine (Ado)-A1R recognition, preactivation of A1R, and A1R-G protein recognition, in hundreds of nanoseconds of simulations. The binding of Ado to the extracellular side of A1R initiates conformational changes and the preactivation of A1R. In turn, the binding of Gi2 to the intracellular side of A1R causes a decrease in the volume of the extracellular orthosteric site and stabilizes the binding of Ado to A1R. Su-GaMD could be a useful tool to reconstruct or even predict ligand-protein and protein-protein recognition pathways on a short timescale. The intermediate states revealed in this study could provide more detailed complementary structural characterizations to facilitate the drug design of A1R in the future.

Lack of Interactions Between Alteplase/Tenecteplase and the Adenosine A1R/A3R Agonist AST-004.

BACKGROUND: AST-004, a small molecule agonist of the adenosine A1 and A3 receptors, is a potential cerebroprotectant for patients with acute stroke and is currently in clinical trials. Drug-drug interactions are critically important to assess in the context of acute stroke care. Lytic therapy with tPA (tissue-type plasminogen activator)-induced plasmin formation (alteplase) is the only available pharmacotherapy for acute stroke. Consequently, it is imperative to evaluate potential interactions between AST-004 and tPAs such as alteplase and tenecteplase. METHODS: The interactions between AST-004 and tPAs were evaluated in 3 ways in preparation for AST-004 phase II trials. First, the metabolic stability of AST-004 was determined in the presence of alteplase and plasmin. Second, the potential for AST-004 to influence the thrombolytic efficacy of alteplase and tenecteplase was evaluated with an in vitro assay system utilizing a fluorogenic substrate of plasmin. Finally, the potential for AST-004 to influence the thrombolytic efficacy of alteplase was also determined with an in vitro thrombolysis assay of human blood thrombi. RESULTS: Neither alteplase nor plasmin affected the stability of AST-004 in vitro. In 2 different in vitro systems, AST-004 had no effect on the ability of alteplase or tenecteplase to generate plasmin, and AST-004 had no effect on the thrombolytic efficacy of alteplase to lyse blood clots in human blood. CONCLUSIONS: These studies indicate that there will be no interactions between AST-004 and tPAs such as alteplase or tenecteplase in patients with stroke undergoing thrombolytic therapy.

Development of Putative Bivalent Dicovalent Ligands for the Adenosine A1 Receptor.

Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A1R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A1R of an A1R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A1R antagonists and showed evidence of covalent binding and simultaneous binding across an A1R homodimer. Greater selectivity for A1R over the adenosine A3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand.

Extracellular cAMP elicits contraction of rat vas deferens: Involvement of ecto-5'-nucleotidase and adenosine A1 receptors.

AIMS: It is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called "extracellular cAMP-adenosine pathway". Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. MAIN METHODS: The effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. KEY FINDINGS: While 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5'-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SIGNIFICANCE: Our data show that activation of ß-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.

Cordycepin suppresses steroidogenic acute regulatory protein expression by reducing SP1 in human granulosa-lutein cells.

In brief: Cordycepin (COR), a compound derived from Cordyceps, is recognized as an adenosine analog with numerous beneficial effects on human health. However, its impact on steroidogenic acute regulatory protein (STAR) expression in ovarian granulosa cells is not well understood. This study demonstrates that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. Abstract: Cordycepin (COR), a pure compound of Cordyceps, is known as an adenosine analog that exerts many beneficial effects on human health. The steroidogenesis mediated by ovarian granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (STAR) regulates the rate-limiting step in steroidogenesis. COR has been shown to stimulate STAR expression in mouse Leydig cells, the steroidogenic cells in the testes. However, the effect of COR on STAR expression in ovarian granulosa cells remains undetermined. In the present study, we show that treatment with COR downregulates STAR expression in a steroidogenic human granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization. We used specific adenosine receptor (AR) antagonists, and our results reveal that the inhibitory effect of COR on STAR expression is mediated by AR-A1, AR-A2A, and AR-A3. In both KGN and primary hGL cells, COR activates ERK1/2 and AKT signaling pathways, but only activation of ERK1/2 is required for the COR-induced downregulation of STAR expression. In addition, our results demonstrate that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. These results provide a better understanding of the biological function of COR on STAR expression in the ovary, which may lead to the development of alternative therapeutic approaches for female reproductive disorders.

Structure of the adenosine-bound human adenosine A1 receptor-Gi complex.

The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.

Putative Role of Adenosine A1 Receptors in Exogenous Ketone Supplements-Evoked Anti-Epileptic Effect.

Approximately 30% of patients with epilepsy are drug-refractory. There is an urgent need to elucidate the exact pathophysiology of different types of epilepsies and the mechanisms of action of both antiseizure medication and metabolic therapies to treat patients more effectively and safely. For example, it has been demonstrated that exogenous ketone supplement (EKS)-generated therapeutic ketosis, as a metabolic therapy, may decrease epileptic activity in both animal models and humans, but its exact mechanism of action is unknown. However, it was demonstrated that therapeutic ketosis, among others, can increase adenosine level, which may enhance activity of A1 adenosine receptors (A1Rs) in the brain. It has also been demonstrated previously that adenosine has anti-epileptic effect through A1Rs in different models of epilepsies. Thus, it is possible that (i) therapeutic ketosis generated by the administration of EKSs may exert its anti-epileptic effect through, among other mechanisms, increased adenosine level and A1R activity and that (ii) the enhanced activity of A1Rs may be a necessary anti-epileptic mechanism evoked by EKS administration-generated ketosis. Moreover, EKSs can evoke and maintain ketosis without severe side effects. These results also suggest that the therapeutic application of EKS-generated ketosis may be a promising opportunity to treat different types of epilepsies. In this literature review, we specifically focus on the putative role of A1Rs in the anti-epileptic effect of EKS-induced ketosis.

The Bifunctional Dimer Caffeine-Indan Attenuates α-Synuclein Misfolding, Neurodegeneration and Behavioral Deficits after Chronic Stimulation of Adenosine A1 Receptors.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson's disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood-brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-ß-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein "loop" conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson's disease therapy.

Dopamine Release in Nucleus Accumbens Is under Tonic Inhibition by Adenosine A1 Receptors Regulated by Astrocytic ENT1 and Dysregulated by Ethanol.

Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor nitrobenzylthioinosine reduced dopamine release and promoted A1R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor [GPCR-activation based (GRAB)-Ado], we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mm) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.SIGNIFICANCE STATEMENT Dopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A1 receptors (A1Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A1Rs on dopamine axons inhibit dopamine release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease.

Cancer-related somatic mutations alter adenosine A1 receptor pharmacology-A focus on mutations in the loops and C-terminus.

G protein-coupled receptors (GPCRs) are known to be involved in tumor progression and metastasis. The adenosine A1 receptor (A1 AR) has been detected to be over-expressed in various cancer cell lines. However, the role of A1 AR in tumor development is not yet well characterized. A series of A1 AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. In this study, we have investigated the pharmacology of mutations located outside of the 7-transmembrane domain by using a "single-GPCR-one-G protein" yeast system. Concentration-growth curves were obtained with the full agonist CPA for 12 mutant receptors and compared to the wild-type hA1 AR. Most mutations located at the extracellular loops (EL) reduced the levels of constitutive activity of the receptor and agonist potency. For mutants at the intracellular loops (ILs) of the receptor, an increased constitutive activity was found for mutant receptor L211R5.69 , while a decreased constitutive activity and agonist response were found for mutant receptor L113F34.51 . Lastly, mutations identified on the C-terminus did not significantly influence the pharmacological function of the receptor. A selection of mutations was also investigated in a mammalian system. Overall, similar effects on receptor activation compared to the yeast system were found with mutations located at the EL, but some contradictory effects were observed for mutations located at the IL. Taken together, this study will enrich the insight of A1 AR structure and function, enlightening the consequences of these mutations in cancer. Ultimately, this may provide potential precision medicine in cancer treatment.

The Agonist of Adenosine A1 Receptor Induced Desensitization of delta Opioid receptor-mediated Raf-1/MEK/ERK Signaling by Feedback Phosphorylation of Raf-1-Ser289/296/301.

Our previous study found that activation of adenosine A1 receptor (A1R) induced phosphorylation of delta opioid receptor (DOR) and desensitization of its downstream signaling molecules, cAMP and Akt. To further investigate the effect of A1R agonist on DOR signaling and the underlying mechanism, we examined the effect of A1R activation upon binding of its agonist N6-cyclohexyl-adenosine (CHA) on DOR-mediated Raf-1/MEK/ERK activation, and found that prolonged CHA exposure resulted in downregulation of DOR-mediated Raf-1/MEK/ERK signaling pathway. CHA-treatment time dependently attenuated Raf-1-Ser338 phosphorylation induced by [D-Pen2,5] enkephalin (DPDPE), a specific agonist of DOR, and further caused downregulation of the Raf-1/MEK/ERK signaling pathway activated by DOR agonist. Moreover, CHA exposure time-dependently induced the phosphorylation of Raf-1-Ser289/296/301, the inhibitory phosphorylation sites that were regulated by negative feedback, thereby inhibiting activation of the MEK/ERK pathway, and this effect could be blocked by MEK inhibitor U0126. Finally, we proved that the heterologous desensitization of the Raf-1/MEK/ERK cascade was essential in the regulation of anti-nociceptive effect of DOR agonists by confirming that such effect was inhibited by pretreatment of CHA. Therefore, we conclude that the activation of A1R inhibits DOR-mediated MAPK signaling pathway via heterologous desensitization of the Raf-1/MEK/ERK cascade, which is a result of ERK-mediated Raf-1-Ser289/296/301 phosphorylation mediated by activation of A1R.

Loss of astrocytic A1 adenosine receptor is involved in a chemotherapeutic agent-induced rodent model of neuropathic pain.

INTRODUCTION/AIMS: Oxaliplatin is a commonly used platinum chemotherapy drug, whereas peripheral neurotoxicity is a widely observed adverse reaction lacking a satisfactory therapeutic strategy. Different adenosine receptors underlying the common neuropathic phenotype play different roles through varied pathophysiological mechanisms. In this study, we investigated the role of adenosine receptor A1 (A1R) in oxaliplatin-induced neuropathic pain and its potential use in an effective therapeutic strategy. METHODS: We established an oxaliplatin-induced neuropathic pain model simulating the mode of chemotherapy administration and observed the related neuropathic behavioral phenotype and implicated mechanisms. RESULTS: Five weekly injections of oxaliplatin for 2 weeks induced a severe and persistent neuropathic pain phenotype in mice. A1R expression in the spinal dorsal horn decreased during this process. Pharmacological intervention against A1R verified its importance in this process. Mechanistically, the loss of A1R expression was mainly attributed to its decreased expression in astrocytes. Consistent with the pharmacological results, the oxaliplatin-induced neuropathic pain phenotype was blocked by specific therapeutic interventions of A1R in astrocytes via lentiviral vectors, and the expression of glutamate metabolism-related proteins was upregulated. Neuropathic pain can be alleviated by pharmacological or astrocytic interventions via this pathway. DISCUSSION: These data reveal a specific adenosine receptor signaling pathway involved in oxaliplatin-induced peripheral neuropathic pain, which is related to the suppression of the astrocyte A1R signaling pathway. This may provide new opportunities for the treatment and management of neuropathic pain observed during oxaliplatin chemotherapy.

In silico identification of A1 agonists and A2a inhibitors in pain based on molecular docking strategies and dynamics simulations.

Most recently, the adenosine is considered as one of the most promising targets for treating pain, with few side effects. It exists in the central nervous system, and plays a key role in nociceptive afferent pathway. It is reported that the A1 receptor (A1R) could inhibit Ca2+ channels to reduce the pain like analgesic mechanism of morphine. And, A2a receptor (A2aR) was reported to enhance the accumulation of AMP (cAMP) and released peptides from sensory neurons, resulting in constitutive activation of pain. Much evidence showed that A1R and A2aR could be served as the interesting targets for the treatment of pain. Herein, virtual screening was utilized to identify the small molecule compounds towards A1R and A2aR, and top six molecules were considered as candidates via amber scores. The molecular dynamic (MD) simulations and molecular mechanics/generalized born surface area (MM/GBSA) were employed to further analyze the affinity and binding stability of the six molecules towards A1R and A2aR. Moreover, energy decomposition analysis showed significant residues in A1R and A2aR, including His1383, Phe1276, and Glu1277. It provided basics for discovery of novel agonists and antagonists. Finally, the agonists of A1R (ZINC19943625, ZINC13555217, and ZINC04698406) and inhibitors of A2aR (ZINC19370372, ZINC20176051, and ZINC57263068) were successfully recognized. Taken together, our discovered small molecules may serve as the promising candidate agents for future pain research.

The antidepressant-like effect of guanosine involves the modulation of adenosine A1 and A2A receptors.

Guanosine has been considered a promising candidate for antidepressant responses, but if this nucleoside could modulate adenosine A1 (A1R) and A2A (A2AR) receptors to exert antidepressant-like actions remains to be elucidated. This study investigated the role of A1R and A2AR in the antidepressant-like response of guanosine in the mouse tail suspension test and molecular interactions between guanosine and A1R and A2AR by docking analysis. The acute (60 min) administration of guanosine (0.05 mg/kg, p.o.) significantly decreased the immobility time in the tail suspension test, without affecting the locomotor performance in the open-field test, suggesting an antidepressant-like effect. This behavioral response was paralleled with increased A1R and reduced A2AR immunocontent in the hippocampus, but not in the prefrontal cortex, of mice. Guanosine-mediated antidepressant-like effect was not altered by the pretreatment with caffeine (3 mg/kg, i.p., a non-selective adenosine A1R/A2AR antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX - 2 mg/kg, i.p., a selective adenosine A1R antagonist), or 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385 - 1 mg/kg, i.p., a selective adenosine A2AR antagonist). However, the antidepressant-like response of guanosine was completely abolished by adenosine (0.5 mg/kg, i.p., a non-selective adenosine A1R/A2AR agonist), N-6-cyclohexyladenosine (CHA - 0.05 mg/kg, i.p., a selective adenosine A1 receptor agonist), and N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA - 0.1 mg/kg, i.p., a selective adenosine A2A receptor agonist). Finally, docking analysis also indicated that guanosine might interact with A1R and A2AR at the adenosine binding site. Overall, this study reinforces the antidepressant-like of guanosine and unveils a previously unexplored modulation of the modulation of A1R and A2AR in its antidepressant-like effect.

The impact of inosine on hippocampal synaptic transmission and plasticity involves the release of adenosine through equilibrative nucleoside transporters rather than the direct activation of adenosine receptors.

Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30 µM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300 µM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) µM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300 µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100 µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37 µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.

Comparative Study of Receptor-, Receptor State-, and Membrane-Dependent Cholesterol Binding Sites in A2A and A1 Adenosine Receptors Using Coarse-Grained Molecular Dynamics Simulations.

We used coarse-grained molecular dynamics (CG MD) simulations to study protein-cholesterol interactions for different activation states of the A2A adenosine receptor (A2AR) and the A1 adenosine receptor (A1R) and predict new cholesterol binding sites indicating amino acid residues with a high residence time in three biologically relevant membranes. Compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-cholesterol and POPC-phosphatidylinositol-bisphosphate (PIP2)-cholesterol, the plasma mimetic membrane best described the cholesterol binding sites previously detected for the inactive state of A2AR and revealed the binding sites with long-lasting amino acid residues. We observed that using the plasma mimetic membrane and plotting residues with cholesterol residence time ≥2 µs, our CG MD simulations captured most obviously the cholesterol-protein interactions. For the inactive A2AR, we identified one more binding site in which cholesterol is bound to residues with a long residence time compared to the previously detected, for the active A1R, three binding sites, and for the inactive A1R, two binding sites. We calculated that for the active states, cholesterol binds to residues with a much longer residence time compared to the inactive state for both A2AR and A1R. The stability of the identified binding sites to A1R or A2AR with CG MD simulations was additionally investigated with potential of mean force calculations using umbrella sampling. We observed that the binding sites with residues to which cholesterol has a long residence time in A2AR have shallow binding free energy minima compared to the related binding sites in A1R, suggesting a stronger binding for cholesterol to A1R. The differences in binding sites in which cholesterol is stabilized and interacts with residues with a long residence time between active and inactive states of A1R and A2AR can be important for differences in functional activity and orthosteric agonist or antagonist affinity and can be used for the design of allosteric modulators, which can bind through lipid pathways. We observed a stronger binding for cholesterol to A1R (i.e., generally higher association rates) compared to A2AR, which remains to be demonstrated. For the active states, cholesterol binds to residues with much longer residence times compared to the inactive state for both A2AR and A1R. Taken together, binding sites of active A1R may be considered as promising allosteric targets.

Dual A1 and A2A adenosine receptor antagonists, methoxy substituted 2-benzylidene-1-indanone, suppresses intestinal postprandial glucose and attenuates hyperglycaemia in fructose-streptozotocin diabetic rats.

BACKGROUND/AIM: Recent research suggests that adenosine receptors (ARs) influence many of the metabolic abnormalities associated with diabetes. A non-xanthine benzylidene indanone derivative 2-(3,4-dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1 H-inden-1-one (2-BI), has shown to exhibit higher affinity at A1/A2A ARs compared to caffeine. Due to its structural similarity to caffeine, and the established antidiabetic effects of caffeine, the current study was initiated to explore the possible antidiabetic effect of 2-BI. METHODS: The study was designed to assess the antidiabetic effects of several A1 and/or A2A AR antagonists, via intestinal glucose absorption and glucose-lowering effects in fructose-streptozotocin (STZ) induced diabetic rats. Six-week-old male Sprague-Dawley rats were induced with diabetes via fructose and streptozotocin. Rats were treated for 4 weeks with AR antagonists, metformin and pioglitazone, respectively. Non-fasting blood glucose (NFBG) was determined weekly and the oral glucose tolerance test (OGTT) was conducted at the end of the intervention period. RESULTS: Dual A1/A2A AR antagonists (caffeine and 2-BI) decreased glucose absorption in the intestinal membrane significantly (p < 0.01), while the selective A2A AR antagonist (Istradefylline), showed the highest significant (p < 0.001) reduction in intestinal glucose absorption. The selective A1 antagonist (DPCPX) had the least significant (p < 0.05) reduction in glucose absorption. Similarly, dual A1/A2A AR antagonists and selective A2A AR antagonists significantly reduced non-fast blood glucose and improved glucose tolerance in diabetic rats from the first week of the treatment. Conversely, the selective A1 AR antagonist did not reduce non-fast blood glucose significantly until the 4th week of treatment. 2-BI, caffeine and istradefylline compared well with standard antidiabetic treatments, metformin and pioglitazone, and in some cases performed even better. CONCLUSION: 2-BI exhibited good antidiabetic activity by reducing intestinal postprandial glucose absorption and improving glucose tolerance in a diabetic animal model. The dual antagonism of A1/A2A ARs presents a positive synergism that could provide a new possibility for the treatment of diabetes.

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells.

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.