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International audience; Extracellular adenosine plays prominent roles in the brain in both physiological and pathological conditions. Adenosine can be generated following the degradation of extracellular nucleotides by various types of ectonucleotidases. Several ectonucleotidases are present in the brain parenchyma: ecto-nucleotide triphosphate diphosphohydrolases 1 and 3 (NTPDase 1 and 3), ecto-nucleotide pyrophosphatase/ phosphodiesterase 1 (NPP 1), ecto-5’-nucleotidase (eN), and tissue non-specific alkaline phosphatase (TNAP, whose function in the brain has received little attention). Here we examined, in a living brain preparation, the role of these ectonucleotidases in generating extracellular adenosine. We recorded local field potentials evoked by electrical stimulation of the lateral olfactory tract in the mouse piriform cortex in vitro. Variations in adenosine level were evaluated by measuring changes in presynaptic inhibition generated by adenosine A1 receptors (A1Rs) activation. A1R-mediated presynaptic inhibition was present endogenously and was enhanced by bath-applied AMP and ATP. We hypothesized that inhibiting ectonucleotidases would reduce extracellular adenosine concentration, which would result in a weakening of presynaptic inhibition. However, inhibiting TNAP had no effect in controlling endogenous adenosine action and no effect on presynaptic inhibition induced by bath-applied AMP. Furthermore, contrary to our expectation, inhibiting TNAP reinforced, rather than reduced, presynaptic inhibition induced by bath-applied ATP. Similarly, inhibition of NTPDase 1 and 3, NPP1, and eN induced stronger, rather than weaker presynaptic inhibition, both in endogenous condition and with bath-applied ATP and AMP. Consequently, attempts to suppress the functions of extracellular adenosine by blocking its extracellular synthesis in living brain tissue could have functional impacts opposite to those anticipated.

The adenosine A

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

The primary cause of harmaline tremor, which is a model of essential tremor (ET) in animals, is excessive activation of olivocerebellar glutamatergic climbing fibers. Our recent study indicated that 5′-chloro-5′-deoxy-(±)-N6-(±)-(endo-norborn-2-yl)adenosine (5′Cl5′d-(±)-ENBA), a potent and selective adenosine A1 receptor (A1) agonist, inhibited harmaline tremor. The present study was aimed to evaluate the role of glutamatergic transmission system in 5′Cl5′d-(±)-ENBA tremorolytic action in the harmaline model in rats, by analyzing glutamate release in the motor nuclei of the thalamus and mRNA expression of glutamatergic neuron markers (vGlut1/2) in reference to the general neuronal activity marker (zif-268) in different brain structures. The extracellular glutamate level in the motor thalamus was evaluated by in vivo microdialysis and the vGlut1/vGlut2 and zif-268 mRNA expression was analyzed by in situ hybridization. The intensity of tremor was measured automatically using Force Plate Actimeters (FPAs). 5′Cl5′d-(±)-ENBA (0.5 mg/kg) given 30 min before harmaline (30 mg/kg) decreased the harmaline-induced excessive glutamate release in the motor thalamus and reversed harmaline - induced molecular effects, such as elevation of the vGlut1 mRNA expression in the inferior olive (IO) and decrease in the motor cortex, as well as an increase of the zif-268 mRNA expression in the IO, motor thalamus and motor cortex. Moreover, 5′Cl5′d-(±)-ENBA reduced harmaline tremor by lowering its power in 9–15 Hz frequency band. Our findings show that A1 stimulation decreases glutamate release in the motor thalamic nuclei in the harmaline model of ET, suggesting that A1 receptors, especially in this structure, may be a potential therapeutic target in this disorder.

Background Disturbances of dopaminergic and glutamatergic transmissions have been suggested to be involved in the pathomechanisms underlying psychotic symptoms of schizophrenia. In line with this concept, hyperlocomotion induced by the dopaminomimetic amphetamine and the uncompetitive antagonist of NMDA receptors MK-801 (dizocilpine) in rodents is a generally established model for screening of new potential antipsychotic drugs. Since recent studies have indicated that receptors for adenosine may be targets for antipsychotic therapy, the aim of the present study was to investigate an influence of 5′-Cl-5′-deoxy-ENBA, a potent and selective adenosine A1 receptor agonist, on hyperlocomotion induced by amphetamine and MK-801. Methods Locomotor activity was measured by Force Plate Actimeters where four force transducers located below the corners of the floor of the cage tracked the animal position on a Cartesian plane at each time point. Results Hyperlocomotion induced by either amphetamine (1 mg/kg sc) or MK-801 (0.3 mg/kg ip) was inhibited by 5′-Cl-5′-deoxy-ENBA (0.1 mg/kg ip). The effect of 5′-Cl-5′-deoxy-ENBA on the amphetamine- and MK-801-induced hyperlocomotion was antagonized by the selective antagonist of adenosine A1 receptor DPCPX at doses of 1 and 2 mg/kg ip, respectively. Conclusion The present study suggests that stimulation of adenosine A1 receptors may produce antipsychotic effects.

Background and purpose Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A1 , A2A , A2B , and A3 ). We have investigated the effect of two A1 -receptor-selective agonists and the novel A1 -receptor bitopic ligand VCP746 on the rat cardiovascular system. Experimental approach The regional haemodynamic responses of these agonist was investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal, mesenteric arteries and the descending abdominal aorta and the jugular vein and caudal artery catheterized. Cardiovascular responses were measured following intravenous infusion (3 min each dose) of CCPA (120, 400, and 1,200 ng·kg-1 ·min-1 ), capadenoson or adenosine (30, 100, and 300 μg·kg-1 ·min-1 ), or VCP746 (6, 20, and 60 μg·kg-1 ·min-1 ) following pre-dosing with DPCPX (0.1 mg·kg-1 , i.v.) or vehicle. Key results CCPA produced a significant A1 -receptor-mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A1 -receptor-mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A1 -receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A2A - and A2B -receptors. Conclusions and implications These results suggest VCP746 mediates its cardiovascular effects via activation of A2 rather than A1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A1 allosteric ligand moieties.

Background and Purpose: Treatment with A1R/A3R (adenosine A1 and A3 receptor) agonists in rodent models of acute ischemic stroke results in significantly reduced lesion volume, indicating activation of adenosine A1R or A3R is cerebroprotective. However, dosing and timing required for cerebroprotection has yet to be established, and whether adenosine A1R/A3R activation will lead to cerebroprotection in a gyrencephalic species has yet to be determined. Methods: The current study used clinical study intervention timelines in a nonhuman primate model of transient, 4-hour middle cerebral artery occlusion to investigate a potential cerebroprotective effect of the dual adenosine A1R/A3R agonist AST-004. Bolus and then 22 hours intravenous infusion of AST-004 was initiated 2 hours after transient middle cerebral artery occlusion. Primary outcome measures included lesion volume, lesion growth kinetics, penumbra volume as well as initial pharmacokinetic-pharmacodynamic relationships measured up to 5 days after transient middle cerebral artery occlusion. Secondary outcome measures included physiological parameters and neurological function. Results: Administration of AST-004 resulted in rapid and statistically significant decreases in lesion growth rate and total lesion volume. In addition, penumbra volume decline over time was significantly less under AST-004 treatment compared with vehicle treatment. These changes correlated with unbound AST-004 concentrations in the plasma and cerebrospinal fluid as well as estimated brain A1R and A3R occupancy. No relevant changes in physiological parameters were observed during AST-004 treatment. Conclusions: These findings suggest that administration of AST-004 and combined A1R/A3R agonism in the brain are efficacious pharmacological interventions in acute ischemic stroke and warrant further clinical evaluation.

Adenosine A1 receptor (A1R) activation, stimulating lipogenesis and decreasing insulin resistance, could be useful for metabolic syndrome management in obese subjects. Since full A1R agonists induce harmful side-effects, while partial agonists show a better pharmacological profile, we investigated the influence of two derivatives of the full A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA), C1 and C2 behaving as A1R partial agonists in animal models, on the adipogenic differentiation of stromal/stem cells (ASCs) from human subcutaneous adipose tissue, which mainly contribute to increase fat mass in obesity. The ASCs from normal-weight subjects showed increased proliferation and A1R expression but reduced adipogenic differentiation compared to obese individual-derived ASCs. Cell exposure to CCPA, C1, C2 or DPCPX, an A1R antagonist, did not affect ASC proliferation, while mainly C2 and DPCPX significantly decreased adipogenic differentiation of both ASC types, reducing the activity of glycerol-3-phosphate dehydrogenase and the expression of PPARγ and FABP-4, all adipogenic markers, and phosphorylation of Akt in the phosphatidylinositol-3-kinase pathway, which plays a key-role in adipogenesis. While requiring confirmation in in vivo models, our results suggest that A1R partial agonists or antagonists, by limiting ASC differentiation into adipocytes and, thereby, fat mass expansion, could favor development/worsening of metabolic syndrome in obese subjects without a dietary control.

Hepatic ischemia/reperfusion injury (IRI) is aggravated by steatosis and is a main risk factor in fatty liver transplantation. Adenosine receptors (ARs) are emerging as therapeutic targets in liver diseases. By using cellular and in vivo systems of hepatic steatosis and IRI, here we evaluated the effects of pharmacological A2AR and A1R activation. The A2AR agonist CGS21680 protected the primary steatotic murine hepatocyte from IR damage and the activation of ASK1 and JNK. Such an effect was attributed to a phosphatidylinositol-3-kinase (PI3K)/Akt-dependent inhibition of ASK1. By contrast, the A1R agonist CCPA enhanced IR damage, intracellular steatosis and oxidative species (OS) production, thereby further increasing the lipid/OS-dependent ASK1-JNK stimulation. The CGS2680 and CCPA effects were nullified by a genetic ASK1 downregulation in steatotic hepatoma C1C7 cells. In steatotic mice livers, CGS21680 protected against hepatic IRI and ASK1/JNK activation whereas CCPA aggravated hepatic steatosis and IRI, and enhanced ASK1 and JNK stimulation. These results evidence a novel mechanism of CGS21680-mediated hepatoprotection, i.e., the PI3K/AKT-dependent inhibition of ASK1, and they show that CGS21680 and CCPA reduces and enhances the IRI of fatty liver, respectively, by preventing or increasing the activation of the cytotoxic ASK1/JNK axis. They also indicate the selective employment of A2AR agonists as an effective therapeutic strategy to prevent IRI in human fatty liver surgery.

Insomnia, the most common sleep disorder, is characterized by a longer sleep latency, greater sleep fragmentation, and consequent excessive daytime fatigue. Due to the various side effects of prescribed hypnotics, demand for new drugs is still high. Recent studies have suggested the adenosine receptor (AR) as a potential therapeutic target for insomnia, however, clinically useful hypnotics targeting AR are not yet available. In the present study, we evaluated the hypnotic effect of rosmarinic acid, a phenolic compound widely found in medicinal plants, through pentobarbital-induced sleep test, electroencephalography/electromyography (EEG/EMG), and immunohistochemistry in mice. The underlying mechanisms were assessed by pharmacological approach using 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and SCH5826, antagonists for A

Peripheral A1 adenosine receptor signaling has been shown to have analgesic effects in a variety of pain conditions. However, it is not yet fully elucidated for the precise molecular mechanisms. Acid sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons responding to protons. Given that both A1 adenosine receptors and ASICs are present in dorsal root ganglia (DRG) neurons, we therefore investigated whether there was a cross-talk between the two types of receptors. Herein, electrophysiological recordings showed that the A1 adenosine receptor agonist N

Sevoflurane affects on the A1 receptor in the central nervous system and potentiates the action of neuromuscular blocking agents. In the present study, we investigated whether sevoflurane (SEVO) has the ability to potentiate the neuromuscular blocking effect of rocuronium and if the specific antagonist of adenosine receptor (SLV320) can reverse this effect. In this study, phrenic nerve–hemidiaphragm tissue specimens were obtained from 40 Sprague–Dawley (SD) rats. The specimens were immersed in an organ bath filled with Krebs buffer and stimulated by a train‐of‐four (TOF) pattern using indirect supramaximal stimulation at 20 s intervals. The specimens were randomly allocated to control, 2‐chloroadenosine (CADO), SEVO, or SLV320 + SEVO groups. In the CADO and SLV320 + SEVO groups, CADO and SLV320 were added to the organ bath from the start to a concentration of 10 μM and 10 nM, respectively. We then proceeded with rocuronium‐induced blockade of >95% depression of the first twitch tension of TOF (T1) and TOF ratio (TOFR). In the SEVO and SLV320 + SEVO groups, SEVO was added to the Krebs buffer solution to concentration of 400–500 μM for 10 min. Sugammadex‐induced T1 and TOFR recovery was monitored for 30 min until >95% of T1 and >0.9 of TOFR were confirmed, and the recovery pattern was compared by plotting these data. T1 recovery in the SEVO and CADO groups was significantly delayed compared with the control and SLV320 + SEVO groups (p, Effects of sevoflurane and adenosine receptor antagonist on the sugammadex‐induced recovery from rocuronium‐induced neuromuscular blockade in rodent phrenic nerve‐hemidiaphragm tissue specimens.

Striatal adenosine A(1) receptor (A(1)R) activation can inhibit dopamine release. A(1)Rs 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 A(1)Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A(1)R 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, A(1)R 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 A(1)R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A(1)R-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 A(1)R-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 A(1) receptors (A(1)Rs), 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 A(1)Rs 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.

Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein-coupled receptor (GPCR) function. The adenosine A1 receptor (A1AR) is one of the adenosine receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A1AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A1AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A1AR internalization. These responses were inhibited by the A1AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A1 receptor antagonist (CA200645). The agonist potencies for inducing A1AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT-A1AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A1AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase, which results in the accumulation of acetylcholine and widespread excitotoxic seizure activity. Because current medical countermeasures (anticholinergics, AChE reactivators, and benzodiazepines) lack sufficient anti-seizure efficacy when treatment is delayed, those intoxicated are at risk for severe brain damage or death if treatment is not immediately available. Toward developing a more effective anti-seizure treatment for NA intoxication, this study evaluated the efficacy of A1 adenosine (ADO) receptor (A1AR) agonists in a rat soman seizure model. One minute after exposure to soman (1.6 × LD50, subcutaneous), rats were treated intraperitoneally with one of the following agonists at increasing dose levels until anti-seizure efficacy was achieved: N6-cyclopentaladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), and (±)-5'-chloro-5′-deoxy-ENBA (ENBA). All A1AR agonists were efficacious in preventing seizure and promoting survival. The effective doses for the A1AR agonists were 60 mg/kg CPA, 36 mg/kg CCPA, and 62 mg/kg ENBA. Whereas vehicle-treated rats experienced 100% seizure and 21% survival (N = 28), ADO treatments reduced seizure occurrence and improved survival rates: 8% seizure and 83% survival with CPA (60 mg/kg, N = 12), 17% seizure and 75% survival with CCPA (36 mg/kg, N = 12), and 8% seizure, 83% survival with ENBA (62 mg/kg, N = 12). The brains of ADO-treated rats were also protected from damage as indicated by neurohistopathological analysis. While all ADO agonists provided neuroprotection, rats receiving CCPA and ENBA experienced less severe ADO-induced side effects (e.g., sedation, hypothermia, bradycardia) than with CPA. The data from this study suggest that the ADO signaling pathway is a promising mechanism for countering seizure activity induced by NAs.

(N)-Methanocarba ([3.1.0]bicyclohexyl) adenosines and corresponding ribosides were synthesized to identify novel A1 adenosine receptor (A1AR) agonists for CNS or peripheral applications. Human and mouse AR binding was determined to assess the constrained ring system’s A1AR compatibility. N6-Dicyclobutylmethyl ribose agonist (9, MRS7469, >2000-fold selective for A1AR) and known truncated N6-dicyclopropylmethyl methanocarba 7 (MRS5474) were drug-like. The pure diastereoisomer of known riboside 4 displayed high hA1AR selectivity. Methanocarba modification reduced A1AR selectivity of N6-dicyclopropylmethyl and endo-norbornyladenosines but increased ribavirin selectivity. Most analogues tested (ip) were inactive or weak in inducing mouse hypothermia, despite mA1AR full agonism and variable mA3AR efficacy, but strong hypothermia by 9 depended on A1AR, which reflects CNS activity (determined using A1AR or A3AR null mice). Conserved hA1AR interactions were preserved in modeling of 9 and methanocarba equivalent 24...

Structural studies of a positive allosteric modulator of the adenosine A1 receptor demonstrate the mechanisms by which stabilization of the GPCR–G protein complex bound to its endogenous agonist yields analgesic efficacy in an animal model of neuropathic pain.

Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03–1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain.

Alzheimer’s, Parkinson’s, and multiple sclerosis are neurodegenerative diseases relatedby neuronal degeneration and death in specific areas of the central nervous system. These pathologiesare associated with neuroinflammation, which is involved in disease progression, and haltingthis process represents a potential therapeutic strategy. Evidence suggests that microglia functionis regulated by A1 and A2A adenosine receptors (AR), which are considered as neuroprotective andneurodegenerative receptors, respectively. The manuscript’s aim is to elucidate the role of thesereceptors in neuroinflammation modulation through potent and selective A1AR agonists (N6-cyclopentyl-2′- or 3′-deoxyadenosine substituted or unsubstituted in 2 position) and A2AAR antagonists(9-ethyl-adenine substituted in 8 and/or in 2 position), synthesized in house, using N13 microglialcells. In addition, the combined therapy of A1AR agonists and A2AAR antagonists to modulate neuroinflammationwas evaluated. Results showed that A1AR agonists were able, to varying degrees,to prevent the inflammatory effect induced by cytokine cocktail (tumor necrosis factor (TNF)-α,interleukin (IL)-1β, and interferon (IFN)-γ), while A2AAR antagonists showed a good ability to counteractneuroinflammation. Moreover, the effect achieved by combining the two most effective compounds(1 and 6) in doses previously found to be non-effective was greater than the treatment effectof each of the two compounds used separately at maximal dose.

Metaplasticity is referred to adjustment in the requirements for induction of synaptic plasticity based on the prior history of activity. Synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), has been considered to be the neural processes underlying learning and memory. Previous observations that cordycepin (an adenosine derivative) improved learning and memory seemed to be contradictory to the findings that cordycepin inhibited LTP. Therefore, we speculated that the conflicting reports of cordycepin might be related to metaplasticity. In the current study, population spike (PS) in hippocampal CA1 area of rats was recorded by using electrophysiological method in vivo. The results showed that cordycepin reduced PS amplitude in hippocampal CA1 with a concentration-dependent relationship, and high frequency stimulation (HFS) failed to induce LTP when cordycepin was intrahippocampally administrated but improved LTP magnitude when cordycepin was pre-treated. Cordycepin increased LTD induced by activating N-Methyl-D-aspartate (NMDA) receptors and subsequently facilitated LTP induced by HFS. Furthermore, we found that 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptors antagonist, could block the roles of cordycepin on LTD and LTP. Collectively, cordycepin was able to modulate metaplasticity in hippocampal CA1 area of rats through adenosine A1 receptors. These findings would be helpful to reconcile the conflicting reports in the literatures and provided new insights into the mechanisms underlying cognitive function promotions with cordycepin treatment.

Adenosine acts as an endogenous anticonvulsant and seizure terminator in the brain. Many of its anticonvulsive effects are mediated through the activation of the adenosine A1 receptor, a G protein-coupled receptor with a wide array of targets. Activating A1 receptors is an effective approach to suppress seizures. This review gives an overview of the neuronal targets of the adenosine A1 receptor focusing in particular on signaling pathways resulting in neuronal inhibition. These include direct interactions of G protein subunits, the adenyl cyclase pathway and the phospholipase C pathway, which all mediate neuronal hyperpolarization and suppression of synaptic transmission. Additionally, the contribution of the guanyl cyclase and mitogen-activated protein kinase cascades to the seizure-suppressing effects of A1 receptor activation are discussed. This review ends with the cautionary note that chronic activation of the A1 receptor might have detrimental effects, which will need to be avoided when pursuing A1 receptor-based epilepsy therapies.

Peripheral tramadol's delivery in the temporomandibular joint (TMJ) leads to significant analgesic outcomes and inflammatory process's resolvent actions. Mechanistically, these properties are apart from the opioid system. Nevertheless, the molecular mechanisms behind these effects are still unclear. Therefore, the present study investigated the hypothesis that adenosine A

N-methyl D-aspartate (NMDA) administered at subtoxic dose plays a protective role against neuronal excitotoxicity, a mechanism described as preconditioning. Since the activation of adenosinergic receptors influences the achievement of NMDA preconditioning in the hippocampus, we evaluated the potential functional interplay between adenosine A

The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 μmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 μg/paw significantly reduced glutamate-induced nociception (p0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 μg/paw) significantly blocked the anti-nociceptive effect of CHA (1 μg/paw) (p0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y(1) receptor (P2Y(1)R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A(3) and A(1)ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y(1)R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A

Adenosine is widely known as a potent modulator of innate and acquired immunity. It is released during transplants, and acts on four subtype receptors. In previous studies, we demonstrated that pharmacological preconditioning (PPC), pre-administration of the selective A1 receptor (A1R) agonist led to A1R desensitization, is followed by upregulation of the adenosine A2A receptor. This immunosuppressive effect resulted in lymphopenia, and it reduced T-cell reactivity. The aim of the current study was to challenge the immunosuppressive effects of A1R-PPC in models of allogeneic grafts. PPC mice were treated by intraperitoneal injection using specific adenosine A1R agonist 24 h and 12 h before starting any procedure. We challenged our method in novel allogeneic muscle and skin grafts models. Mice and grafts were assessed by complete blood counts, MLR from PPC splenocytes, and pathological evaluation. We found a significant reduction in WBC and lymphocyte counts in PPC-treated mice. Two-way MLR with splenocytes from PPC grafted mice showed decreased proliferation and anergy. Histology of PPC allogeneic grafts revealed profoundly less infiltration and even less muscle necrosis compared to vehicle treated allografts. Similar results observed in PPC skin transplantation. To conclude, PPC moderated graft rejection in separate allogeneic challenges, and reduced lymphocytes infiltration and ischemic damage.

We have recently demonstrated that N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, acts centrally to induce a visceral antinociception. Since serotonin (5-HT), cannabinoid (CB), dopamine or opioid signaling in the central nervous system is involved in the regulation of visceral sensation, we made a hypothesis that the signaling may play a role in the CPA-induced visceral antinociception. Visceral sensation was evaluated by colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneously administered CPA significantly increased the threshold of colonic distension-induced AWR. Intracisternal injection of either 5-HT

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A1receptors (A1Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K+conductance mediated by Kir4.1/5.1 channels to mediate K+recycling and to set up a favorable driving force for Na+/K+exchange ( 47 ). Intercalated cells express the Cl−ClC-K2/b channel mediating transcellular Cl−reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on Kir4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A1R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A1R agonist N6-cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and Kir4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A1Rs. We propose that inhibition of transcellular Cl−reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.

Glycine receptor α1

Among still comparatively few G protein-coupled receptors, the adenosine A2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A1 receptor (A1AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A2AAR and the A3AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A3AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Extracellular adenosine, a danger signal, can cause hypothermia. We generated mice lacking neuronal adenosine A1 receptors (A1AR, encoded by the Adora1 gene) to examine the contribution of these receptors to hypothermia. Intracerebroventricular injection of the selective A1AR agonist (Cl-ENBA, 5'-chloro-5'-deoxy-N6-endo-norbornyladenosine) produced hypothermia, which was reduced in mice with deletion of A1AR in neurons. A non-brain penetrant A1AR agonist [SPA, N6-(p-sulfophenyl) adenosine] also caused hypothermia, in wild type but not mice lacking neuronal A1AR, suggesting that peripheral neuronal A1AR can also cause hypothermia. Mice expressing Cre recombinase from the Adora1 locus were generated to investigate the role of specific cell populations in body temperature regulation. Chemogenetic activation of Adora1-Cre-expressing cells in the preoptic area did not change body temperature. In contrast, activation of Adora1-Cre-expressing dorsomedial hypothalamus cells increased core body temperature, concordant with agonism at the endogenous inhibitory A1AR causing hypothermia. These results suggest that A1AR agonism causes hypothermia via two distinct mechanisms: brain neuronal A1AR and A1AR on neurons outside the blood-brain barrier. The variety of mechanisms that adenosine can use to induce hypothermia underscores the importance of hypothermia in the mouse response to major metabolic stress or injury.

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

Critical periods of synaptic plasticity facilitate the reordering and refining of neural connections during development, allowing the definitive synaptic circuits responsible for correct adult physiology to be established. Presynaptic spike timing-dependent long-term depression (t-LTD) exists in the hippocampus, which depends on the activation of NMDARs and that probably fulfills a role in synaptic refinement. This t-LTD is present until the third postnatal week in mice, disappearing in the fourth week of postnatal development. We were interested in the mechanisms underlying this maturation related loss of t-LTD and we found that at CA3–CA1 synapses, presynaptic NMDA receptors (pre-NMDARs) are tonically active between P13 and P21, mediating an increase in glutamate release during this critical period of plasticity. Conversely, at the end of this critical period (P22–P30) and coinciding with the loss of t-LTD, these pre-NMDARs are no longer tonically active. Using immunogold electron microscopy, we demonstrated the existence of pre-NMDARs at Schaffer collateral synaptic boutons, where a decrease in the number of pre-NMDARs during development coincides with the loss of both tonic pre-NMDAR activation and t-LTD. Interestingly, this t-LTD can be completely recovered by antagonizing adenosine type 1 receptors (A1R), which also recovers the tonic activation of pre-NMDARs at P22–P30. By contrast, the induction of t-LTD was prevented at P13–P21 by an agonist of A1R, as was tonic pre-NMDAR activation. Furthermore, we found that the adenosine that mediated the loss of t-LTD during the fourth week of development is supplied by astrocytes. These results provide direct evidence for the mechanism that closes the window of plasticity associated with t-LTD, revealing novel events probably involved in synaptic remodeling during development.

Caffeine is widely used to reduce sedation and increase alertness. However, long-term caffeine use may disrupt circadian (daily, 24-h) rhythms and thereby negatively affect health. Here, we examined the effect of caffeine on photic regulation of circadian activity rhythms in mice. We found that entrainment to a standard 12-h light, 12-h dark (LD) photocycle was delayed during oral self-administration of caffeine. Both acute, high-dose caffeine and chronic, oral caffeine exposure potentiated photic phase-delays in mice, suggesting a possible mechanism by which entrainment to LD was delayed. The effect of caffeine on photic phase-resetting was mimicked by administration of adenosine A1, but not A2A, receptor antagonist in mice. Our results support the hypothesis that caffeine interferes with the ability of the circadian clock to respond normally to light.

Among candidate neuroprotective agents, adenosine is thought to be a possible treatment for central nervous system disorders. Adenosine elicits biological effects through four G protein-coupled receptors (A1, A2A, A2B, and A3). The A2A and A2B receptors stimulate adenylyl cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels, whereas A1 and A3 receptors inhibit AC and decrease cAMP levels. Several studies have investigated the effects of adenosine receptors (AdoRs) in glaucoma, because modulation of A1, A2A, or A3 receptor regulates intraocular pressure. In addition, AdoR-related phenomena may induce neuroprotective effects in retinal neurons. Notably, A1, A2A, and A3 receptor agonists reportedly inhibit retinal ganglion cell (RGC) death in in vitro and in vivo glaucoma models. However, there is limited knowledge of the effects of AdoR activation on neurite outgrowth or the regeneration of RGCs. In this report, we described the role of an AdoR subtype in neurite outgrowth and RGC axonal regeneration. The distribution of AdoRs in the retina was evaluated by immunohistochemical analysis. Using primary cultured rat RGCs in vitro and an optic nerve crush model in vivo, neurite elongation was evaluated after stimulation by the following AdoR agonists: CHA, an A1 receptor agonist; CGS21680, an A2A receptor agonist; BAY60-6583, an A2B receptor agonist; and 2-Cl-IB-MECA, an A3 receptor agonist. To determine the mechanism of neurite promotion, the candidate molecules of signal transduction associated with the neurite elongation of AdoRs were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. All four AdoRs (A1, A2A, A2B, and A3) were present in the inner retinal layers. Among the agonists for AdoR, only 2-Cl-IB-MECA significantly promoted neurite outgrowth in primary cultured RGCs. Signaling pathway analyses showed that 2-Cl-IB-MECA caused upregulated phosphorylation of Akt in cultured RGCs. Additionally, LY294002, an inhibitor of Akt, suppressed the neurite-promoting effects of the A3 receptor agonist in RGCs. Moreover, 2-Cl-IB-MECA increased the number of regenerating axons in the optic nerve crush model. Taken together, these data indicate that activation of the A3 receptor, not the A1 or A2 receptors, promotes in vitro and in vivo neurite outgrowth during the regeneration of rat RGCs, which is caused by the activation of an Akt-dependent signaling pathway. Therefore, AdoR activation may be a promising candidate for the development of novel regenerative modalities for glaucoma and other optic neuropathies.

Central adenosine A1-receptor (A1AR)-mediated signals play a role in the induction of hibernation. We determined whether activation of the central A1AR enables rats to maintain normal sinus rhythm even after their body temperature has decreased to less than 20 °C. Intracerebroventricular injection of an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), followed by cooling decreased the body temperature of rats to less than 20 °C. Normal sinus rhythm was fundamentally maintained during the extreme hypothermia. In contrast, forced induction of hypothermia by cooling anesthetized rats caused cardiac arrest. Additional administration of pentobarbital to rats in which hypothermia was induced by CHA also caused cardiac arrest, suggesting that the operation of some beneficial mechanisms that are not activated under anesthesia may be essential to keep heart beat under the hypothermia. These results suggest that central A1AR-mediated signals in the absence of anesthetics would provide an appropriate condition for maintaining normal sinus rhythm during extreme hypothermia.

Mice enter bouts of daily torpor, drastically reducing metabolic rate, core body temperature (T b), and heart rate (HR), in response to reduced caloric intake. Because central adenosine activation has been shown to induce a torpor-like state in the arctic ground squirrel, and blocking the adenosine-1 (A1) receptor prevents daily torpor, we hypothesized that central activation of the A1 adenosine receptors would induce a bout of natural torpor in mice. To test the hypothesis, mice were subjected to four different hypothermia bouts: natural torpor, forced hypothermia (FH), isoflurane-anesthesia, and an intracerebroventricular injection of the selective A1 receptor agonist N6-cyclohexyladenosine (CHA). All conditions induced profound hypothermia. T b fell more rapidly in the FH, isoflurane-anesthesia, and CHA conditions compared to torpor, while mice treated with CHA recovered at half the rate of torpid mice. FH, isoflurane-anesthesia, and CHA-treated mice exhibited a diminished drop in HR during entry into hypothermia as compared to torpor. Mice in all conditions except CHA shivered while recovering from hypothermia, and only FH mice shivered substantially while entering hypothermia. Circulating lactate during the hypothermic bouts was not significantly different between the CHA and torpor conditions, both of which had lower than baseline lactate levels. Arrhythmias were largely absent in the FH and isoflurane-anesthesia conditions, while skipped beats were observed in natural torpor and periodic extended (>1 s) HR pauses in the CHA condition. Lastly, the hypothermic bouts showed distinct patterns of gene expression, with torpor characterized by elevated hepatic and cardiac Txnip expression and all other hypothermic states characterized by elevated c-Fos and Egr-1 expression. We conclude that CHA-induced hypothermia and natural torpor are largely different physiological states.

AIM: The aim of this study was to examine the role of adenosine A(1) receptors in the harmaline‐induced tremor in rats using 5′‐chloro‐5′‐deoxy‐(±)‐ENBA (5′Cl5′d‐(±)‐ENBA), a brain‐penetrant, potent, and selective adenosine A(1) receptor agonist. METHODS: Harmaline was injected at a dose of 15 mg/kg ip and tremor was measured automatically in force‐plate actimeters by an increased averaged power in the frequency band of 9‐15 Hz (AP2) and by tremor index (a difference in power between AP2 and averaged power in the frequency band of 0‐8 Hz). The zif‐268 mRNA expression was additionally analyzed by in situ hybridization in several brain structures. RESULTS: 5′Cl5′d‐(±)‐ENBA (0.05‐0.5 mg/kg ip) dose dependently reduced the harmaline‐induced tremor and this effect was reversed by 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective antagonist of adenosine A(1) receptors (1 mg/kg ip). Harmaline increased the zif‐268 mRNA expression in the inferior olive, cerebellar cortex, ventroanterior/ventrolateral thalamic nuclei, and motor cortex. 5′Cl5′d‐(±)‐ENBA reversed these increases in all the above structures. DPCPX reduced the effect of 5′Cl5′d‐(±)‐ENBA on zif‐268 mRNA in the motor cortex. CONCLUSION: This study suggests that adenosine A(1) receptors may be a potential target for the treatment of essential tremor.

Accumulating evidence suggests that the anti-diabetic drug, metformin, exerts anti-proliferative effects in many types of cancers. However, the function and mechanisms of metformin in human colorectal cancer (CRC) remain unknown. Here, we show that metformin induces growth inhibition and apoptosis through activating AMPK-mTOR pathway in human colorectal cancer cells. Notably, metformin treatment significantly up-regulated adenosine A1 receptor (ADORA1) expression in human colorectal cancer cells, while suppression of ADORA1 activity by its specific inhibitor rescued the growth inhibition induced by metformin. Moreover, ADORA1-mediated growth inhibition and apoptosis induced by metformin is AMPK-mTOR pathway dependent in human colorectal cancer cells. Taken together, these results indicate that metformin suppresses human colorectal cancer growth by inducing apoptosis via ADORA1, which provide evidence the anti-neoplastic effects of metformin in the treatment of human colorectal cancer.

Aims: Neladenoson bialanate is a partial adenosine A1 receptor agonist with demonstrated beneficial effects on cardiac function in animal models. We aimed to assess the dose–response effect of neladenoson bialanate on cardiac structure and function, clinical outcome, and safety in patients with heart failure (HF) with reduced ejection fraction (HFrEF). Methods and results: PANTHEON was a dose-finding, phase IIb, randomized, double-blind, placebo-controlled trial conducted in 92 centres in 11 countries including 462 patients with chronic HFrEF, randomized to once daily oral dose of neladenoson bialanate (5, 10, 20, 30, and 40 mg) or placebo. The primary endpoints were change from baseline to 20 weeks in left ventricular ejection fraction (LVEF) (echocardiography) and in N-terminal pro-B-type natriuretic peptide (NT-proBNP). Mean age of the patients was 67 years, 17% were female, mean LVEF was 28%, mean NT-proBNP was 2085 ng/L. After 20 weeks of treatment, there was no dose–effect of neladenoson bialanate on changes in NT-proBNP or LVEF (primary endpoints). No effect of neladenoson bialanate was found on left ventricular volumes, high-sensitivity troponin T, or cardiovascular mortality, HF hospitalization, and urgent visits for HF (secondary endpoints). There was a dose-dependent increase in creatinine and cystatin C, and a dose-dependent decrease in estimated glomerular filtration rate and heart rate. Conclusions: In patients with chronic HFrEF, treatment with neladenoson bialanate was not associated with dose-dependent favourable effects on cardiac structure and function, cardiac risk markers, or clinical outcome but was associated with a dose-dependent decrease in renal function. Clinical Trial Registration: ClinicalTrials.gov Identifier NCT02992288.

Opioids are effective analgaesic agents, but serious adverse effects such as tolerance and withdrawal contribute to opioid dependence and limit their use. Opioid withdrawal is a common occurrence in human opiate addicts that is not life-threatening. Studies have shown that the mesocorticolimbic system, especially the nucleus accumbens, is an important region in drug addiction and adenosine receptors play a modulatory role in the mechanism of action of drug dependence and withdrawal. The aim of this study was to investigate the effects of the selective A1 receptor agonist CPA (N6 -cyclopentyladenosine) on withdrawal symptoms, and the concentration of dopamine and noradrenaline in the nucleus accumbens and locomotor activity behaviour during naloxone-precipitated withdrawal in morphine-dependent rats. The local administration of CPA (1.5, 3.0, and 6.0 mmol/L bilateral 250 nL) into the nucleus accumbens decreased the Gellert-Holtzman withdrawal scale, and increased concentrations of dopamine and noradrenaline in the same region during naloxone-induced withdrawal. Our findings suggest that administration of the A1 receptor agonist significantly decreased withdrawal behaviours and increased dopamine and noradrenaline concentrations in opioid withdrawal in a dose-dependent manner. These results demonstrate that adenosine receptors should be examined as a potential mechanism that could be exploited for the treatment of morphine withdrawal.

AIMS: Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. MAIN METHODS: Dogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10 μM ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). KEY FINDINGS: A1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (p

Objective To observe the effect of adenosine A1 receptor in the hippocampus of mice on GSK-3β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. Method The model of middle cerebral artery occlusion (MCAO) was established and grouped into electroacupuncture pretreatment group (EA group), MCAO group, and sham-operated group (Sham group). The neurobehavioral manifestation, the volume of cerebral infarction, and its related protein changes in mice in each group were observed. Then, adenosine Α1 receptor antagonist and agonist were injected intraperitoneally to observe the effects of A1 receptor on the phosphorylation level of GSK-3β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. Results (1) Compared with the MCAO group (24 hours after reperfusion), the infarct size in the EA group decreased significantly, and the Garcia neurological score and phosphorylation level of GSK-3β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. Conclusions Electroacupuncture pretreatment can increase GSK-3β phosphorylation level via activating A1 receptor, to protect neurons in ischemia-reperfusion injury.β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury.

Ethnopharmacological relevance Ginkgo biloba leaves and Panax ginseng are Chinese medicine commonly used in combination for cerebral disease. Aim of the study To investigate the effect of standard extract of Ginkgo biloba leaves (EGb) on facilitating brain uptake of ginsenoside and its underlying mechanisms. Materials and methods The increasing uptake of ginsenosides in the brain of rats by EGb were detected by LC–MS/MS analysis. Evans blue and FITC-dextran leakage were determined to evaluate blood-brain barrier (BBB) permeability in vivo. Transendothelial electrical resistance (TEER) and Na–F penetration rate were measured with a co-culture of the human cerebral microvascular endothelial cell line (hCMEC/D3) and human normal glial cell line (HEB) in vitro BBB model. WB were used to analyzed the expression of BBB tight junctions (TJs) related protein (ZO-1, Occludin, Claudin-3, p-ERM, and p-MLC), ultrastructure of TJs was determined by transmission electron microscope. Results LC–MS/MS analysis demonstrated that EGb could improve brain uptake of ginsenoside Rg1, Re, Rd and Rb1. In vivo study showed that, BBB permeability was significantly increased after EGb administration, evidenced by the markedly increased penetration of FITC-dextran and Evans Blue into the mice brain parenchyma. In the in vitro BBB model, reduced TEER and increased Na–F penetration rate was observed in EGb group, which was associated with alteration of TJs ultrastructure. Furthermore, the expression of p-ERM and p-MLC in hCMEC/D3 as well as mice brain microvessels were significantly upregulated, but no significant change on the expression of TJs proteins (ZO-1, Occludin and Claudin-3). Moreover, the effect of EGb on in vitro BBB permeability and ERM, MLC phosphorylation was counteracted by DPCPX, an A1 adenosine receptor (A1R) antagonist. Conclusions EGb might induce ERM/MLC phosphorylation and increase the cell-cell junction gaps to cause a reversible increase of the BBB permeability via A1R signaling pathway. Our results may contribute to better use of EGb in the treatment of brain diseases.