8-Bicycloalkyl-CPFPX derivatives as potent and selective tools for in vivo imaging of the A1 adenosine receptor.

Imaging of the A1 adenosine receptor (A1R) by positron emission tomography (PET) with 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propyl-xanthine ([18F]CPFPX) has been widely used in preclinical and clinical studies. However, this radioligand suffers from rapid peripheral metabolism and subsequent accumulation of radiometabolites in the vascular compartment. In the present work, we prepared four derivatives of CPFPX by replacement of the cyclopentyl group with norbornane moieties. These derivatives were evaluated by competition binding studies, microsomal stability assays and LC-MS analysis of microsomal metabolites. In addition, the 18F-labeled isotopologue of 8-(1-norbornyl)-3-(3-fluoropropyl)-1-propylxanthine (1-NBX) as the most promising candidate was prepared by radiofluorination of the corresponding tosylate precursor and the resulting radioligand ([18F]1-NBX) was evaluated by permeability assays with Caco-2 cells and in vitro autoradiography in rat brain slices. Our results demonstrate that 1-NBX exhibits significantly improved A1R affinity and selectivity when compared to CPFPX and that it does not give rise to lipophilic metabolites expected to cross the blood-brain-barrier in microsomal assays. Furthermore, [18F]1-NBX showed a high passive permeability (Pc = 6.9 ± 2.9 × 10-5 cm/s) and in vitro autoradiography with this radioligand resulted in a distribution pattern matching A1R expression in the brain. Moreover, a low degree of non-specific binding (5%) was observed. Taken together, these findings identify [18F]1-NBX as a promising candidate for further preclinical evaluation as potential PET tracer for A1R imaging.

Activation of hepatic adenosine A1 receptor ameliorates MASH via inhibiting SREBPs maturation.

Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved clinical drugs. Adenosine A1 receptor (A1R), belonging to the G-protein-coupled receptors (GPCRs) superfamily, is mainly distributed in the central nervous system and major peripheral organs with wide-ranging physiological functions; however, the exact role of hepatic A1R in MAFLD remains unclear. Here, we report that liver-specific depletion of A1R aggravates while overexpression attenuates diet-induced metabolic-associated fatty liver (MAFL)/MASH in mice. Mechanistically, activation of hepatic A1R promotes the competitive binding of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) to sequestosome 1 (SQSTM1), rather than protein kinase A (PKA) leading to SCAP degradation in lysosomes. Reduced SCAP hinders SREBP1c/2 maturation and thus suppresses de novo lipogenesis and inflammation. Higher hepatic A1R expression is observed in patients with MAFL/MASH and high-fat diet (HFD)-fed mice, which is supposed to be a physiologically adaptive response because A1R agonists attenuate MAFL/MASH in an A1R-dependent manner. These results highlight that hepatic A1R is a potential target for MAFL/MASH therapy.

Elucidating the role of intrinsic adenosine A1 receptors in acute alcoholism using human-induced pluripotent stem cell-derived hepatocytes.

Acute alcoholic hepatitis (AAH) from binge drinking is a serious disease. It is associated with a high mortality rate, especially among young adults. Apoptosis is known to be a primary cause of liver damage, and it can be induced by either intrinsic signaling pathways or by reactive oxygen species (ROS). Adenosine A1 receptors (ADORA1) are known to be involved in ethanol metabolism; however, underlying mechanism is not well understood. For investigating how the intrinsic ADORA1 function in ethanol metabolism in normal human hepatocytes without interference by extrinsic molecules, primary hepatocytes pose a challenge, due to unavoidable contamination by other kinds of cells in the liver. Also, they are difficult to culture stably. As a novel alternative, hepatocytes derived from human-induced pluripotent stem cells were employed because they display similar function to primary hepatocytes and they can be stably cultured. The dynamics and integrity of signal transduction mechanisms were investigated by following chronological changes in gene expression. This shed light on how and when the ADORA1 function and on causal relationships between the pathways and clinical symptoms. The findings of the present study shows that ADORA1 are most activated soon after exposure to ethanol, and transfection of small interfering RNA targeting ADORA1-messenger-RNA (ADORA1-siRNA) into the hepatocytes significantly suppresses production of actin protein and ROS. It suggests that ADORA1 in the liver contribute to apoptosis in acute alcoholism through both intrinsic pathway and ROS activity. Also, actin that is abundant in the cells could be an appropriate biomarker evaluating hepatic function status.

Caffeine Impaired Acupuncture Analgesia in Inflammatory Pain by Blocking Adenosine A1 Receptor.

Caffeine consumption inhibits acupuncture analgesic effects by blocking adenosine signaling. However, existing evidence remains controversial. Hence, this study aimed to examine the adenosine A1 receptor (A1R) role in moderate-dose caffeine-induced abolishing effect on acupuncture analgesia using A1R knockout mice (A1R-/-). We assessed the role of A1R in physiological sensory perception and its interaction with caffeine by measuring mechanical and thermal pain thresholds and administering A1R and adenosine 2A receptor antagonists in wild-type (WT) and A1R-/- mice. Formalin- and complete Freund's adjuvant (CFA)-induced inflammatory pain models were recruited to explore moderate-dose caffeine effect on pain perception and acupuncture analgesia in WT and A1R-/- mice. Moreover, a C-fiber reflex electromyogram in the biceps femoris was conducted to validate the role of A1R in the caffeine-induced blockade of acupuncture analgesia. We found that A1R was dispensable for physiological sensory perception and formalin- and CFA-induced hypersensitivity. However, genetic deletion of A1R impaired the antinociceptive effect of acupuncture in A1R-/- mice under physiological or inflammatory pain conditions. Acute moderate-dose caffeine administration induced mechanical and thermal hyperalgesia under physiological conditions but not in formalin- and CFA-induced inflammatory pain. Moreover, caffeine significantly inhibited electroacupuncture (EA) analgesia in physiological and inflammatory pain in WT mice, comparable to that of A1R antagonists. Conversely, A1R deletion impaired the EA analgesic effect and decreased the caffeine-induced inhibitory effect on EA analgesia in physiological conditions and inflammatory pain. Moderate-dose caffeine administration diminished the EA-induced antinociceptive effect by blocking A1R. Overall, our study suggested that caffeine consumption should be avoided during acupuncture treatment. PERSPECTIVE: Moderate-dose caffeine injection attenuated EA-induced antinociceptive effect in formalin- and CFA-induced inflammatory pain mice models by blocking A1R. This highlights the importance of monitoring caffeine intake during acupuncture treatment.

Adenosine A1 receptors of the medullary solitary tract arbitrate the nicotine counteraction of neuroinflammation and cardiovascular dysfunction in septic rats.

The cholinergic pathway plays a crucial role in improving inflammatory end-organ damage. Given the interplay between cholinergic and adenosinergic neurotransmission, we tested the hypothesis that central adenosine A1 receptors (A1ARs) modulate the nicotine counteraction of cardiovascular and inflammatory insults induced by sepsis in rats. Sepsis was induced by cecal ligation and puncture (CLP) 24-h before cardiovascular measurements. Nicotine (25-100 µg/kg i.v.) dose-dependently reversed septic manifestations of hypotension and impaired heart rate variability (HRV) and cardiac sympathovagal balance. Like nicotine, intracisternal (i.c.) administration of N(6)-cyclopentyladenosine (CPA, A1AR agonist) to CLP rats increased indices of HRV and sympathovagal balance. Moreover, greater surges in these parameters were noted upon simultaneous nicotine/CPA administration. The favorable influences of nicotine on blood pressure and HRV in sepsis were diminished after central blockade of A1ARs by i.c. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX). Molecular studies revealed that (i) septic rises in myocardial and brainstem nucleus of solitary tract (NTS) NFκB expression were abrogated by nicotine and largely reinstated after blockade of A1ARs, and (ii) A1AR expression in the same areas was reduced by DPCPX. It is concluded that myocardial and medullary A1ARs facilitate the cholinergic counteraction of cardiac and neuroinflammation induced by sepsis and interrelated cardiomyopathic and neuropathic hitches.

Activation of adenosine A1 receptor potentiates metabotropic glutamate receptor 1-mediated Ca2+ mobilization in the rat hippocampal marginal zone.

Two subtypes of group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, participate in the regulation of cell excitability and synaptic plasticity in the central nervous system. They couple to the Gq/11 protein and release Ca2+ from the intracellular stores. In the marginal zone of the neonatal hippocampus, Cajal-Retzius (CR) cells, which control radial migration of neurons, express the subtype mGluR1. The adenosine A1 receptor (A1R) is also G-protein coupled and is extensively expressed in the central nervous system. The interactions among G-protein-coupled receptors have been predicted previously, however, there is insufficient evidence of functional interactions between naturally occurring receptors. In this study, potentiation of the mGluR1-mediated response by A1R activation was demonstrated in hippocampal CR cells. Fluorescence imaging revealed that the application of A1R agonists intensified mGluR1-induced elevation of intracellular Ca2+ concentration ([Ca2+]i). Activation of A1R did not change [Ca2+]i. The potentiated responses were independent of extracellular Ca2+ and prevented by the Gi inhibitor. The potentiation of mGluR1-induced [Ca2+]i. elevation was also enhanced by mGluR2/3 activation. These results suggest that mGluR1 and A1R cooperatively influence postnatal hippocampal development by facilitating Ca2+ mobilization in CR cells.

Small molecule allosteric modulation of the adenosine A1 receptor.

G protein-coupled receptors (GPCRs) represent the target for approximately a third of FDA-approved small molecule drugs. The adenosine A1 receptor (A1R), one of four adenosine GPCR subtypes, has important (patho)physiological roles in humans. A1R has well-established roles in the regulation of the cardiovascular and nervous systems, where it has been identified as a potential therapeutic target for a number of conditions, including cardiac ischemia-reperfusion injury, cognition, epilepsy, and neuropathic pain. A1R small molecule drugs, typically orthosteric ligands, have undergone clinical trials. To date, none have progressed into the clinic, predominantly due to dose-limiting unwanted effects. The development of A1R allosteric modulators that target a topographically distinct binding site represent a promising approach to overcome current limitations. Pharmacological parameters of allosteric ligands, including affinity, efficacy and cooperativity, can be optimized to regulate A1R activity with high subtype, spatial and temporal selectivity. This review aims to offer insights into the A1R as a potential therapeutic target and highlight recent advances in the structural understanding of A1R allosteric modulation.

Protective effects of caffeine against palmitate-induced lipid toxicity in primary rat hepatocytes is associated with modulation of adenosine receptor A1 signaling.

BACKGROUND: Epidemiological evidence has shown an association between coffee consumption and reduced risk for chronic liver diseases, including metabolic-dysfunction-associated liver disease (MALFD). Lipotoxicity is a key cause of hepatocyte injury during MAFLD. The coffee component caffeine is known to modulate adenosine receptor signaling via the antagonism of adenosine receptors. The involvement of these receptors in the prevention of hepatic lipotoxicity has not yet been explored. The aim of this study was to explore whether caffeine protects against palmitate-induced lipotoxicity by modulating adenosine receptor signaling. METHODS: Primary hepatocytes were isolated from male rats. Hepatocytes were treated with palmitate with or without caffeine or 1,7DMX. Lipotoxicity was verified using Sytox viability staining and mitochondrial JC-10 staining. PKA activation was verified by Western blotting. Selective (ant)agonists of A1AR (DPCPX and CPA, respectively) and A2AR (istradefyline and regadenoson, respectively), the AMPK inhibitor compound C, and the Protein Kinase A (PKA) inhibitor Rp8CTP were used. Lipid accumulation was verified by ORO and BODIPY 453/50 staining. RESULTS: Caffeine and its metabolite 1,7DMX prevented palmitate-induced toxicity in hepatocytes. The A1AR antagonist DPCPX also prevented lipotoxicity, whereas both the inhibition of PKA and the A1AR agonist CPA (partially) abolished the protective effect. Caffeine and DPCPX increased lipid droplet formation only in palmitate-treated hepatocytes and decreased mitochondrial ROS production. CONCLUSIONS: The protective effect of caffeine against palmitate lipotoxicity was shown to be dependent on A1AR receptor and PKA activation. Antagonism of A1AR also protects against lipotoxicity. Targeting A1AR receptor may be a potential therapeutic intervention with which to treat MAFLD.

Reversal of Tau-Dependent Cognitive Decay by Blocking Adenosine A1 Receptors: Comparison of Transgenic Mouse Models with Different Levels of Tauopathy.

The accumulation of tau is a hallmark of several neurodegenerative diseases and is associated with neuronal hypoactivity and presynaptic dysfunction. Oral administration of the adenosine A1 receptor antagonist rolofylline (KW-3902) has previously been shown to reverse spatial memory deficits and to normalize the basic synaptic transmission in a mouse line expressing full-length pro-aggregant tau (TauΔK) at low levels, with late onset of disease. However, the efficacy of treatment remained to be explored for cases of more aggressive tauopathy. Using a combination of behavioral assays, imaging with several PET-tracers, and analysis of brain tissue, we compared the curative reversal of tau pathology by blocking adenosine A1 receptors in three mouse models expressing different types and levels of tau and tau mutants. We show through positron emission tomography using the tracer [18F]CPFPX (a selective A1 receptor ligand) that intravenous injection of rolofylline effectively blocks A1 receptors in the brain. Moreover, when administered to TauΔK mice, rolofylline can reverse tau pathology and synaptic decay. The beneficial effects are also observed in a line with more aggressive tau pathology, expressing the amyloidogenic repeat domain of tau (TauRDΔK) with higher aggregation propensity. Both models develop a progressive tau pathology with missorting, phosphorylation, accumulation of tau, loss of synapses, and cognitive decline. TauRDΔK causes pronounced neurofibrillary tangle assembly concomitant with neuronal death, whereas TauΔK accumulates only to tau pretangles without overt neuronal loss. A third model tested, the rTg4510 line, has a high expression of mutant TauP301L and hence a very aggressive phenotype starting at ~3 months of age. This line failed to reverse pathology upon rolofylline treatment, consistent with a higher accumulation of tau-specific PET tracers and inflammation. In conclusion, blocking adenosine A1 receptors by rolofylline can reverse pathology if the pathological potential of tau remains below a threshold value that depends on concentration and aggregation propensity.

Structure-based virtual screening discovers potent and selective adenosine A1 receptor antagonists.

Development of subtype-selective leads is essential in drug discovery campaigns targeting G protein-coupled receptors (GPCRs). Herein, a structure-based virtual screening approach to rationally design subtype-selective ligands was applied to the A1 and A2A adenosine receptors (A1R and A2AR). Crystal structures of these closely related subtypes revealed a non-conserved subpocket in the binding sites that could be exploited to identify A1R selective ligands. A library of 4.6 million compounds was screened computationally against both receptors using molecular docking and 20 A1R selective ligands were predicted. Of these, seven antagonized the A1R with micromolar activities and several compounds displayed slight selectivity for this subtype. Twenty-seven analogs of two discovered scaffolds were designed, resulting in antagonists with nanomolar potency and up to 76-fold A1R-selectivity. Our results show the potential of structure-based virtual screening to guide discovery and optimization of subtype-selective ligands, which could facilitate the development of safer drugs.

Activation of the adenosine A1 receptor in the lumbosacral spinal cord improves bladder overactivity in rats with cystitis induced by cyclophosphamide.

PURPOSE: To investigate the effect of intrathecal administration of CCPA, an adenosine A1 receptor agonist, on voiding function in rats with cystitis induced by cyclophosphamide (CYP). METHODS: Thirty 8-week-old Sprague Dawley rats were randomly divided into a control group (n = 15) and a cystitis group (n = 15). Cystitis was induced by a single intraperitoneal injection of CYP (200 mg/kg, dissolved in physiological saline) in rats. Control rats were injected intraperitoneally with physiological saline. The PE10 catheter reached the level of L6-S1 spinal cord through L3-4 intervertebral space for intrathecal injection. Forty-eight hours after intraperitoneal injection, urodynamic tests were conducted to observe the effect of intrathecal administration of 10% dimethylsulfoxide (vehicle) and 1 nmol CCPA on micturition parameters, including basal pressure (BP), threshold pressure (TP), maximal voiding pressure (MVP), intercontraction interval (ICI), voided volume (VV), residual volume (RV), bladder capacity (BC), and voiding efficiency (VE). Histological changes of the bladder of cystitis rats were studied through hematoxylin-eosin staining (HE staining). Moreover, Western blot and immunofluorescence were used to study the expression of adenosine A1 receptor in the L6-S1 dorsal spinal cord in both groups of rats. RESULTS: HE staining revealed submucosal hemorrhage, edema, and inflammatory cell infiltration in the bladder wall of cystitis rats. The urodynamic test showed significant increase in BP, TP, MVP and RV in cystitis rats, while ICI, VV, BC and VE decreased significantly, indicating bladder overactivity. CCPA inhibited the micturition reflex in both control and cystitis rats, and significantly increased TP, ICI, VV, BC, and VE, but had no significant effect on BP, MVP and RV. Western blot and immunofluorescence showed that there was no significant difference in the expression of adenosine A1 receptor in the L6-S1 dorsal spinal cord between the control and cystitis rats. CONCLUSION: The findings of this study suggest that intrathecal administration of the adenosine A1 receptor agonist CCPA alleviates CYP-induced bladder overactivity. Furthermore, our results indicate that the adenosine A1 receptor in the lumbosacral spinal cord may be a promising target for treatment of bladder overactivity.

An allosteric modulator of the adenosine A1 receptor potentiates the antilipolytic effect in rat adipose tissue.

The adenosine A1 receptor plays important roles in tuning free fatty acid (FFA) levels and represents an attractive target for metabolic disorders. Though remarkable progress has been achieved in the exploitation of effective (orthosteric) A1 receptor agonists in modulating aberrant FFA levels, the effect of A1 receptor allosteric modulation on lipid homeostasis is less investigated. Herein we sought to explore the effect of an allosteric modulator on the action of an A1 receptor orthosteric agonist in regulating the lipolytic process in vitro and in vivo. We examined the binding kinetics of a selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) in the absence or presence of an allosteric modulator (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)-phenyl]methanone (PD81,723) on rat adipocyte membranes. We also examined the allosteric effects of PD81,723 on mediating the CCPA-induced inhibition of cAMP accumulation, HSL (hormone-sensitive lipase) phosphorylation and FFA production in in vitro and in vivo models. Our results demonstrated that PD81,723 slowed down the dissociation of CCPA from the A1 receptor, which, consequently, potentiated the antilipolytic action of CCPA through downregulating the cAMP/HSL pathway. Our study exemplified the application of A1 receptor allosteric modulators as an alternative for metabolic disease treatments.

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.

Deep brain stimulation suppresses epileptic seizures in rats via inhibition of adenosine kinase and activation of adenosine A1 receptors.

AIMS: Deep brain stimulation (DBS) of the anterior nucleus of the thalamus, is an effective therapy for patients with drug-resistant epilepsy, yet, its mechanism of action remains elusive. Adenosine kinase (ADK), a key negative regulator of adenosine, is a potential modulator of epileptogenesis. DBS has been shown to increase adenosine levels, which may suppress seizures via A1 receptors (A1 Rs). We investigated whether DBS could halt disease progression and the potential involvement of adenosine mechanisms. METHODS: Control group, SE (status epilepticus) group, SE-DBS group, and SE-sham-DBS group were included in this study. One week after a pilocarpine-induced status epilepticus, rats in the SE-DBS group were treated with DBS for 4 weeks. The rats were monitored by video-EEG. ADK and A1 Rs were tested with histochemistry and western blot, respectively. RESULTS: Compared with the SE group and SE-sham-DBS group, DBS could reduce the frequency of spontaneous recurrent seizures (SRS) and the number of interictal epileptic discharges. The DPCPX, an A1 R antagonist, reversed the effect of DBS on interictal epileptic discharges. In addition, DBS inhibited the overexpression of ADK and the downregulation of A1 Rs. CONCLUSION: The findings indicate that DBS can reduce SRS in epileptic rats via inhibition of ADK and activation of A1 Rs. A1 Rs might be a potential target of DBS for the treatment of epilepsy.

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.

Neuroprotection by trans-resveratrol in rats with N-methyl-D-aspartate (NMDA)-induced retinal injury: Insights into the role of adenosine A1 receptors.

Adenosine A1 receptors (AA1R) have been shown to counteract N-methyl-D-aspartate (NMDA)-mediated glutamatergic excitotoxicity. In the present study, we investigated the role of AA1R in neuroprotection by trans-resveratrol (TR) against NMDA-induced retinal injury. In total, 48 rats were divided into the following four groups: normal rats pretreated with vehicle; rats that received NMDA (NMDA group); rats that received NMDA after pretreatment with TR; and rats that received NMDA after pretreatment with TR and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an AA1R antagonist. Assessment of general and visual behaviour was performed using the open field test and two-chamber mirror test, respectively, on Days 5 and 6 post NMDA injection. Seven days after NMDA injection, animals were euthanized, and eyeballs and optic nerves were harvested for histological parameters, whereas retinae were isolated to determine the redox status and expression of pro- and anti-apoptotic proteins. In the present study, the retinal and optic nerve morphology in the TR group was protected from NMDA-induced excitotoxic damage. These effects were correlated with the lower retinal expression of proapoptotic markers, lipid peroxidation, and markers of nitrosative/oxidative stress. The general and visual behavioural parameters in the TR group showed less anxiety-related behaviour and better visual function than those in the NMDA group. All the findings observed in the TR group were abolished by administration of DPCPX.

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.

The role of adenosine in alcohol-induced respiratory suppression.

Alcohol-related poisoning is the foremost cause of death resulting from excessive acute alcohol consumption. Respiratory failure is crucial to the pathophysiology of fatal alcohol poisoning. Alcohol increases accumulation of extracellular adenosine. Adenosine suppresses breathing. The goal of this investigation was to test the hypothesis that adenosine signaling contributes to alcohol-induced respiratory suppression. In the first experiment, the breathing of mice was monitored following an injection of the non-selective adenosine receptor antagonist caffeine (40 mg/kg), alcohol (5 g/kg), or alcohol and caffeine combined. Caffeine reduced alcohol-induced respiratory suppression suggesting that adenosine contributes to the effects of alcohol on breathing. The second experiment utilized the same experimental design, but with the blood brain barrier impermeant non-selective adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT, 60 mg/kg) instead of caffeine. 8-SPT did not reduce alcohol-induced respiratory suppression suggesting that adenosine is contributing to alcohol-induced respiratory suppression in the central nervous system. The third and fourth experiments used the same experimental design as the first, but with the selective A1 receptor antagonist DPCPX (1 mg/kg) and the selective A2A receptor antagonist istradefylline (3.3 mg/kg). Istradefylline, but not DPCPX, reduced alcohol-induced respiratory suppression indicating an A2A receptor mediated effect. In the fifth experiment, alcohol-induced respiratory suppression was evaluated in Adk+/- mice which have impaired adenosine metabolism. Alcohol-induced respiratory suppression was exacerbated in Adk+/- mice. These findings indicate that adenosinergic signaling contributes to alcohol-induced respiratory suppression. Improving our understanding of how alcohol affects breathing may lead to better treatment strategies and better outcomes for patients with severe alcohol poisoning.

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.

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.