Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity.

The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational analysis of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.

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

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

Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment.

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

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

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

Torpor-like Hypothermia Induced by A1 Adenosine Receptor Agonist: A Novel Approach to Protect against Neuroinflammation.

Hypothermia is a promising clinical therapy for acute injuries, including neural damage, but it also faces practical limitations due to the complexities of the equipment and procedures required. This study investigates the use of the A1 adenosine receptor (A1AR) agonist N6-cyclohexyladenosine (CHA) as a more accessible method to induce steady, torpor-like hypothermic states. Additionally, this study investigates the protective potential of CHA against LPS-induced sepsis and neuroinflammation. Our results reveal that CHA can successfully induce a hypothermic state by activating a neuronal circuit similar to the one that induces physiological torpor. This state is characterized by maintaining a steady core body temperature below 28 °C. We further found that this torpor-like state effectively mitigates neuroinflammation and preserves the integrity of the blood-brain barrier during sepsis, thereby limiting the infiltration of inflammatory factors into the central nervous system. Instead of being a direct effect of CHA, this protective effect is attributed to inhibiting pro-inflammatory responses in macrophages and reducing oxidative stress damage in endothelial cells under systemic hypothermia. These results suggest that A1AR agonists such as CHA could potentially be potent neuroprotective agents against neuroinflammation. They also shed light on possible future directions for the application of hypothermia-based therapies in the treatment of sepsis and other neuroinflammatory conditions.

Adenosine A1R/A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke.

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.

Inhibitors of ectonucleotidases have paradoxical effects on synaptic transmission in the mouse cortex.

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.

Ex Vivo Feedback Control of Neurotransmission Using a Photocaged Adenosine A1 Receptor Agonist.

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.

Functional interplay between adenosine A2A receptor and NMDA preconditioning in fear memory and glutamate uptake in the mice hippocampus.

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 A1 and A2A receptors (A1R and A2AR) activities and NMDA preconditioning. Adult male Swiss mice received saline (NaCl 0.9 g%, i.p.) or a nonconvulsant dose of NMDA (75 mg/kg, i.p.) and 24 h later they were treated with the one of the ligands: A1R agonist (CCPA, 0.2 mg/kg, i.p.) or antagonist (DPCPX, 3 mg/kg, i.p.), A2AR agonist (CGS21680, 0.05 mg/kg, i.p.) or antagonist (ZM241385, 0.1 mg/kg, i.p.) and subjected to contextual fear conditioning task. Binding properties and content of A2AR and glutamate uptake were assessed in the hippocampus of mice subjected to NMDA preconditioning. Treatment with CGS21680 increased the time of freezing during the exposure of animals to the new environment. NMDA preconditioning did not affect the freezing time of mice per se, but it prevented the response observed after the activation of A2AR. Furthermore, the activation of A2AR by CGS21680 after the preconditioning blocked the increase of glutamate uptake induced by NMDA preconditioning. The immunodetection of A2AR in total hippocampal homogenates showed no significant differences evoked by NMDA preconditioning and did not alter A2AR maximum binding for the selective ligand [3H]CGS21680. These results demonstrate changes in A2AR functionality in mice following NMDA preconditioning.

Intramuscularly administered A1 adenosine receptor agonists as delayed treatment for organophosphorus nerve agent-induced Status Epilepticus.

Exposure to organophosphorus nerve agents (NAs) like sarin (GB) and soman (GD) can lead to sustained seizure activity, or status epilepticus (SE). Previous research has shown that activation of A1 adenosine receptors (A1ARs) can inhibit neuronal excitability, which could aid in SE termination. Two A1AR agonists, 2-Chloro-N6-cyclopentyladenosine (CCPA) and N-Bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), were effective in terminating GD-induced SE in rats when administered via intraperitoneal (IP) injection. However, IP injection is not a clinically relevant route of administration. This study evaluated the efficacy of these agonists in terminating NA-induced SE when administered via intramuscular (IM) route. Adult male rats were exposed subcutaneously (SC) to either GB (150 µg/kg) or GD (90 µg/kg) and were treated with ENBA or CCPA at 15, 30, or 60 min after seizure onset or left untreated. Up to 7 days after exposure, deeply anesthetized rats were euthanized and perfused brains were removed for histologic assessment of neuropathology (i.e., neuronal damage) in six brain regions (amygdala, cerebral cortex, piriform cortex, thalamus, dorsal hippocampus, and ventral hippocampus). A total neuropathy score (0-24) was determined for each rat by adding the scores from each of the six regions. The higher the total score the more severe the neuropathology. With the GB model and 60 min treatment delay, ENBA-treated rats experienced 78.6% seizure termination (N = 14) and reduced neuropathology (11.6 ± 2.6, N = 5), CCPA-treated rats experienced 85.7% seizure termination (N = 14) and slightly reduced neuropathology (20.7 ± 1.8, N = 6), and untreated rats experienced no seizure termination (N = 13) and severe neuropathology (22.3 ± 1.0, N = 4). With the GD model and 60 min treatment delay, ENBA-treated rats experienced 92.9% seizure termination (N = 14) and reduced neuropathology (13.96 ± 1.8, N = 9), CCPA-treated rats experienced 78.6% seizure termination (N = 14) and slightly reduced neuropathology (22.0 ± 0.9, N = 10); and untreated rats experienced 16.7% seizure termination (N = 12) and severe neuropathology (22.0 ± 1.8, N = 5). While ENBA and CCPA both demonstrate a clear ability to terminate SE when administered up to 60 min after seizure onset, ENBA offers more neuroprotection, making it a promising candidate for NA-induced SE.

Evaluation of adenosine A1 receptor agonists as neuroprotective countermeasures against Soman intoxication in rats.

Soman, an organophosphorus (OP) compound, disrupts nervous system function through inactivation of acetylcholinesterase (AChE), the enzyme that breaks down acetylcholine at synapses. Left untreated, a state of prolonged seizure activity (status epilepticus, SE) is induced, causing widespread neuronal damage and associated cognitive and behavioral impairments. Previous research demonstrated that therapeutic stimulation of A1 adenosine receptors (A1ARs) can prevent or terminate soman-induced seizure. This study examined the ability of three potent A1AR agonists to provide neuroprotection and, ultimately, prevent observable cognitive and behavioral deficits following exposure to soman. Sprague Dawley rats were challenged with a seizure-inducing dose of soman (1.2 x LD50) and treated 1 min later with one of the following A1AR agonists: (6)-Cyclopentyladenosine (CPA), 2-Chloro-N6-cyclopentyladenosine (CCPA) or N-bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (cdENBA). An active avoidance shuttle box task was used to evaluate locomotor responses to aversive stimuli at 3, 7 and 14 days post-exposure. Animals treated with CPA, CCPA or cdENBA demonstrated a higher number of avoidance responses and a faster reaction to the aversive stimulus than the soman/saline control group across all three sessions. Findings suggest that A1AR agonism is a promising neuroprotective countermeasure, capable of preventing the long-term deficits in learning and memory that are characteristic of soman intoxication.

Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice.

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.

The role of peripheral adenosine receptors in glutamate-induced pain nociceptive behavior.

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 (p<0.01 and p<0.001 vs. vehicle, respectively, n=8-10). In contrast, i.pl. injection of hydrochloride hydrate (CGS21680, an adenosine A2A receptor agonist) (1 µg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 µg/paw) caused a significant reduction (p<0.05, n=7-8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs-8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1-10 µg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1-100 µg/paw), alloxazine (an A2B antagonist, 0.1-3 µg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1-100 µg/paw) (p>0.05 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) (p<0.05, n=7-9). Similarly, ZM241385 (20 µg/paw) administered prior to CGS21680 (1 µg/paw) significantly blocked CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 µg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7-8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 µg/paw) (p<0.05, n=7-9) but not by the A2A antagonist ZM241385 (10 µg/paw, p>0.05). 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.

Combined Therapy of A1AR Agonists and A2AAR Antagonists in Neuroinflammation.

Alzheimer's, Parkinson's, and multiple sclerosis are neurodegenerative diseases related by neuronal degeneration and death in specific areas of the central nervous system. These pathologies are associated with neuroinflammation, which is involved in disease progression, and halting this process represents a potential therapeutic strategy. Evidence suggests that microglia function is regulated by A1 and A2A adenosine receptors (AR), which are considered as neuroprotective and neurodegenerative receptors, respectively. The manuscript's aim is to elucidate the role of these receptors 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 microglial cells. In addition, the combined therapy of A1AR agonists and A2AAR antagonists to modulate neuroinflammation was 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)-1ß, and interferon (IFN)-γ), while A2AAR antagonists showed a good ability to counteract neuroinflammation. 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 effect of each of the two compounds used separately at maximal dose.

Adenosine inhibits the basolateral Cl- ClC-K2/b channel in collecting duct intercalated cells.

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 A1 receptors (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.

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology.

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 P2Y1 receptor (P2Y1R) 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 A3 and A1ARs. 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 P2Y1R 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.

A Taxicab geometry quantification system to evaluate the performance of in silico methods: a case study on adenosine receptors ligands.

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.

The role of adenosine A1 receptor agonist in adenosine augmentation therapy for patients with refractory epilepsy in Sturge-Weber syndrome: An in vitro electrophysiological study.

PURPOSES: This study was to further explore the adenosine dysfunction in refractory epilepsy in Sturge-Weber Syndrome (SWS), to evaluate the neuronal-level effect of the A1 receptor (A1R) agonist on both excitatory pyramidal neurons and inhibitory interneurons, to discuss the possibility of adenosine augmentation therapy (AAT) using A1R agonist for treating refractory epilepsy in SWS. MATERIALS AND METHODS: The intrinsic excitatory properties of pyramidal cells (PCs) and fast-spiking (FS) interneurons from human brain tissues with SWS cases and malformations of cortical development (MCD) cases were compared using electrophysiology. With application of either A1R agonist or antagonist, the neuronal-level effect of A1R agonist was evaluated in vitro in PCs and FS interneurons from SWS cases and MCD cases. RESULTS: No significant difference of passive excitatory properties of PCs and FS interneurons was found between SWS cases and MCD cases. In terms of the neuronal-level effect of A1R agonist, with 22.88 ±â€¯1.12% percentage of decreased frequency, FS interneurons showed relatively highest sensitivity of A1R agonist application, compared with PCs from SWS cases and FS interneurons and PCs from MCD cases. CONCLUSION: Our results supported the potential of AATs using A1R agonist to be a novel therapy for reducing life burden from patients with refractory epilepsy in SWS, with application to epileptic generation region but not propagation region.

Adenosine Receptor-Mediated Developmental Loss of Spike Timing-Dependent Depression in the Hippocampus.

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.

The role of adenosine A1 receptors in the nucleus accumbens during morphine withdrawal.

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.