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.

Learning and memory function preserved by delayed A1 adenosine receptor agonist treatment following soman intoxication in rats and a humanized esterase mouse model.

Exposure to organophosphorus compounds, such as soman (GD), cause widespread toxic effects, sustained status epilepticus, neuropathology, and death. The A1 adenosine receptor agonist N-bicyclo-(2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), when given 1 min after GD exposure, provides neuroprotection and prevents behavioral impairments. Here, we tested the ability of ENBA at delayed treatment times to improve behavioral outcomes via a two-way active avoidance task in two male animal models, each consisting of saline and GD exposure groups. In a rat model, animals received medical treatments (atropine sulfate [A], 2-PAM [P], and midazolam [MDZ]) or AP + MDZ + ENBA at 15 or 30 min after seizure onset and were subjected to behavioral testing for up to 14 days. In a human acetylcholinesterase knock-in serum carboxylesterase knock-out mouse model, animals received AP, AP + MDZ, AP + ENBA, or AP + MDZ + ENBA at 15 min post seizure onset and were subjected to the behavioral task on days 7 and 14. In rats, the GD/AP + MDZ + ENBA group recovered to saline-exposed avoidance levels while the GD/AP + MDZ group did not. In mice, in comparison with GD/AP + MDZ group, the GD/AP + MDZ + ENBA showed decreases in escape latency, response latency, and pre-session crossings, as well as increases in avoidances. In both models, only ENBA-treated groups showed control level inter-trial interval crossings by day 14. Our findings suggest that ENBA, alone and as an adjunct to medical treatments, can improve behavioral and cognitive outcomes when given at delayed time points after GD intoxication.

Hypothermia as potential therapeutic approach to attenuating soman-induced seizure, neuropathology, and mortality with an adenosine A1 receptor agonist and body cooling.

Organophosphorus nerve agents, such as soman (GD), produce excitotoxic effects resulting in sustained status epilepticus (SSE) and brain damage. Previous work shows that neuronal inhibitory effects of A1 adenosine receptor (A1AR) agonists, such as N6- Bicyclo (2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (Cl-ENBA), suppresses GD-induced SSE and improves neuropathology. Some other physiologic effects of these agonists are hypothermia, hypotension, and sedation. Hypothermia may also shield the brain from injury by slowing down chemical insults, lessening inflammation, and contributing to improved neurological outcomes. Therefore, we attempted to isolate the hypothermic effect from ENBA by assessing the neuroprotective efficacy of direct surface body cooling in a rat GD-induced SSE model, and comparing the effects on seizure termination, neuropathology, and survival. Male rats implanted with a body temperature (Tb) transponder and electroencephalographic (EEG) electrodes were primed with asoxime (HI-6), exposed to GD 30 min later, and then treated with Cl-ENBA or had Tb lowered directly via body cooling at 30 min after the onset of seizure activity. Afterwards, they were either allowed to develop hypothermia as expected, or received thermal support to maintain normothermic Tb for a period of 6-h. Neuropathology was assessed at 24 h. Regardless of Cl-ENBA or surface cooling, all hypothermic GD-exposed groups had significantly improved 24-h survival compared to rats with normothermic Tb (81% vs. 39%, p < 0.001). Cl-ENBA offered neuroprotection independently of hypothermic Tb. While hypothermia enhanced the overall efficacy of Cl-ENBA by improving survival outcomes, body cooling didn't reduce seizure activity or neuropathology following GD-induced SSE.

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.

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.

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.

Examining the Role of the Linker in Bitopic N6-Substituted Adenosine Derivatives Acting as Biased Adenosine A1 Receptor Agonists.

The adenosine A1 receptor is a therapeutic target based on its ability to provide cardioprotection during episodes of myocardial ischemia and reperfusion injury. However, the clinical translation of A1R agonists has been hindered by dose-limiting adverse effects (bradycardia and hypotension). Previously, we demonstrated that the bitopic agonist VCP746 (1), consisting of an adenosine pharmacophore linked to an allosteric moiety, can stimulate cardioprotective A1R signaling effects in the absence of unwanted bradycardia. This study maps the structure-activity relationships of 1 through modifications to the linker moiety. Derivatives differing in the flexibility, length, and nature of the linker were assessed, which revealed that the linker is tolerant of several modifications including added rigidity. Ligands featuring 1,4-disubstituted 1,2,3-triazoles were the most biased of the novel analogues but also displayed sub-nanomolar potency in a cAMP accumulation assay at the A2BR. To our knowledge, 10 is the most potent A2BR agonist published to date.

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.

Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine.

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.

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.

Involvement of the adenosine A1 receptor in the hypnotic effect of rosmarinic acid.

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 A1R and A2AR, respectively. Receptor-binding assay and functional agonism were also performed. Our study provides a new evidence that rosmarinic acid has a direct binding activity (Ki = 14.21 ± 0.3 µM) and agonistic activity for A1R. We also found that rosmarinic acid significantly decreased sleep fragmentation and onset latency to NREM sleep, and these effects were abolished by DPCPX. The results from c-Fos immunostaining showed that rosmarinic acid decreased the neuronal activity in wake-promoting brain regions, such as the basal forebrain and the lateral hypothalamus, while increasing the neuronal activity in the ventrolateral preoptic nucleus, a sleep-promoting region; all these effects were significantly inhibited by DPCPX. Taken together, this study suggests that rosmarinic acid possesses novel activity as an A1R agonist and thereby exerts a hypnotic effect, and thus it may serve as a potential therapeutic agent for insomnia through targeting A1R.

Suppression of ASIC activity by the activation of A1 adenosine receptors in rat primary sensory neurons.

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 N6-cyclopentyladenosine (CPA) suppressed acid-induced currents and action potentials, which were mediated by ASICs, in rat DRG neurons. CPA inhibited the maximum response to protons, as shown a downward shift of concentration-response curve for protons. The CPA-induced suppression of ASIC currents was blocked by the A1 adenosine receptor antagonist KW-3902 and also prevented by intracellular application of the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. Finally, intraplantar pretreatment of CPA dose-dependently relieved acid-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed ASICs via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons, which was a novel potential mechanism underlying analgesia of peripheral A1 adenosine receptors.

Multipotent Stromal Cells from Subcutaneous Adipose Tissue of Normal Weight and Obese Subjects: Modulation of Their Adipogenic Differentiation by Adenosine A1 Receptor Ligands.

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.

Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation.

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.

Effects of sevoflurane and adenosine receptor antagonist on the sugammadex-induced recovery from rocuronium-induced neuromuscular blockade in rodent phrenic nerve-hemidiaphragm tissue specimens.

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 < .05). In conclusion, sevoflurane affects on the A1 receptor at the neuromuscular junction and delays sugammadex-induced recovery from neuromuscular blockade.

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 adenosine A1 receptor in the peripheral tramadol's effect in the temporomandibular joint of rats.

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 A1 receptors are involved in the tramadol-induced analgesic and anti-inflammatory effects in the TMJ. Animals were pretreated with an intra-TMJ injection of DPCPX (antagonist of A1 receptor) or tramadol and subsequent nociceptive challenge with an intra-TMJ injection of 1.5% formalin. For over 45 min, the nociceptive behavior was quantitated, and by the end of this assessment, the animals were euthanized, and the periarticular tissue was collected. Lastly, an in vitro assay of BMDM (Bone Marrow-Derived Macrophages) was performed to investigate tramadol activity in macrophages. The intra-TMJ injection of tramadol ameliorates formalin-induced hypernociception along with inhibiting leukocyte migration. The tramadol's peripheral anti-inflammatory effect was mediated by the adenosine A1 receptor and was associated with increased protein expression of α2a-adrenoceptor in the periarticular tissues (p < 0.05: ANOVA, Tukey's test). Also, tramadol inhibits formalin-induced leukocyte migration and protein expression of P2X7 receptors in the periarticular tissue (p < 0.05); however, DPCPX did not alter this effect (p > 0.05). Moreover, DPCPX significantly reduced the protein expression of the M2 macrophage marker, MRC1. In BMDM, tramadol significantly reduces inflammatory cytokines release, and DPCPX abrogated this effect (p < 0.05). We identify tramadol's peripheral effect is mediated by adenosine A1 receptor, possibly expressed in macrophages in the TMJ tissue. We also determined an important discovery related to the activation of A1R/α2a receptors in the tramadol action.