Activation Of Adenosine A1 Receptors Research Articles

Crosstalk Inhibition Between 5-HT2A and Adenosine 2A Receptor-Induced Phrenic Motor Facilitation

Episodic spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of respiratory motor plasticity. Although both intermittent 5-HT2 and 5-HT7 receptor activation elicit pMF, they do so via distinct cellular mechanisms known as the Q (5-HT2A/B) and S (5-HT7) pathways. The Q and S pathways interact via powerful crosstalk inhibition; thus, concurrent activation of both receptor subtypes diminishes/abolishes pMF. At least some of the molecules giving rise to mutually inhibitory interactions between 5-HT2 and 5-HT7 receptors have been identified. Although episodic spinal adenosine 2A (A2A) receptor activation also elicits pMF via the S pathway, we do not know: 1) if 5-HT2 and A2A receptors interact via similar crosstalk inhibition, nor 2) if they do so via the same signaling mechanisms. Thus, we tested the hypothesis that concurrent 5-HT2 and A2A receptor activation diminishes pMF via crosstalk inhibition. Selective 5-HT2A (DOI) and A2A receptor agonists (CGS21680) were delivered to the C4 intrathecal space (3, 6 μl injections, 5-min intervals) in urethane anesthetized, artificially ventilated, vagotomized and paralyzed Sprague Dawley rats. Integrated phrenic nerve burst activity was measured before (baseline), during and 90 minutes post-drug injection. Spinal 5-HT2A and A2A receptor activation both increased phrenic nerve burst amplitude when delivered alone (i.e., pMF); however, pMF was no longer apparent with concurrent receptor activation. The magnitude of pMF, expressed as a % change in phrenic burst amplitude from baseline to 90-min post-injection, was significantly lower with concurrent receptor activation (5-HT2A + A2A= 15 ± 7.7%; n=5) versus 5-HT2A (84 ± 12%; n=5, p=0.0047) and A2A (88 ± 16%; n=5, p=0.0085) alone. Thus, 5-HT2A and A2A receptors interact via crosstalk inhibition similar to 5-HT2 and 5-HT7 receptors. However, the mechanism underlying this crosstalk inhibition remains unknown. NIH HL148030. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spreading Depolarization Induces a Transient Potentiation of Excitatory Synaptic Transmission

Spreading depolarization (SD) is a slowly propagating wave of prolonged activation followed by a period of synaptic suppression. Some prior reports have shown potentiation of synaptic transmission after recovery from synaptic suppression and noted similarities with the phenomenon of long-term potentiation (LTP). Since SD is increasingly recognized as participating in diverse neurological disorders, it is of interest to determine whether SD indeed leads to a generalized and sustained long-term strengthening of synaptic connections. We performed a characterization of SD-induced potentiation, and tested whether distinctive features of SD, including adenosine accumulation and swelling, contribute to reports of SD-induced plasticity. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the hippocampal CA1 subregion of murine brain slices, and SD elicited using focal microinjection of KCl. A single SD was sufficient to induce a consistent potentiation of slope and amplitude of fEPSPs. Both AMPA- and NMDA-receptor mediated components were enhanced. Potentiation peaked ∼20 min after SD recovery and was sustained for ∼30 min. However, fEPSP amplitude and slope decayed over an extended 2-hour recording period and was estimated to reach baseline after ∼3 h. Potentiation was saturated after a single SD and adenosine A1 receptor activation did not mask additional potentiation. Induction of LTP with theta-burst stimulation was not altered by prior induction of SD and molecular mediators known to block LTP induction did not block SD-induced potentiation. Together, these results indicate an intermediate duration potentiation that is distinct from hippocampal LTP and may have implications for circuit function for 1–2 h following SD.

Astrocytes in preoptic area regulate acute nociception-induced hypothermia through adenosine receptors.

The preoptic area (POA) of the hypothalamus, crucial in thermoregulation, has long been implicated in the pain process. However, whether nociceptive stimulation affects body temperature and its mechanism remains poorly studied. We used capsaicin, formalin, and surgery to induce acute nociceptive stimulation and monitored rectal temperature. Optical fiber recording, chemical genetics, confocal imaging, and pharmacology assays were employed to confirm the role and interaction of POA astrocytes and extracellular adenosine. Immunofluorescence was utilized for further validation. Acute nociception could activate POA astrocytes and induce a decrease in body temperature. Manipulation of astrocytes allowed bidirectional control of body temperature. Furthermore, acute nociception and astrocyte activation led to increased extracellular adenosine concentration within the POA. Activation of adenosine A1 or A2A receptors contributed to decreased body temperature, while inhibition of these receptors mitigated the thermo-lowering effect of astrocytes. Our results elucidate the interplay between acute nociception and thermoregulation, specifically highlighting POA astrocyte activation. This enriches our understanding of physiological responses to painful stimuli and contributes to the analysis of the anatomical basis involved in the process.

Seizure and regadenoson: An underestimated concern

For 15 years, regadenoson, a selective adenosine 2A (A2A) receptor agonist, has been largely used as a pharmacologic stress agent in myocardial perfusion studies. It acts by increasing coronary blood flow. It has been shown to be non-inferior to adenosine for the detection of reversible myocardial ischemia. Regadenoson has several advantages, including less serious adverse effects, better tolerance and easier practical administration. However, specific adverse effects on the central nervous system, although rarely reported, can provocoke seizure through A2A receptor activation. We report here a case of regadenoson-associated-seizure and review the relevant literature. A 73-year-old male with a past medical history of hypertension, atrial fibrillation, stroke, hemodialysis and type 2 diabetes was referred for evaluation of hypokinesia of the apical segments. He had an unique episode of epilepsia in 2008. Less than one minute after regadenoson injection, he had a partial tonic-clonic seizure of the right upper and lower limbs, which lasted for 30seconds and resolved spontaneously. This case report is a reminder that this under-diagnosed adverse effect must be taken into consideration when using regadenoson for cardiac stress testing.

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.

Abstract A074: Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway

Abstract Background: The Wnt/β-catenin pathway controls the growth of pancreatic carcinoma by regulating processes such as cell cycle progression, apoptosis, and the tumor immune microenvironment. Treating pancreatic cancer with Wnt pathway inhibitors was found to be toxic in clinical studies. Namodenoson, an A3 adenosine receptor (A3AR) agonist, is under clinical development as a treatment of advanced liver cancer, where namodenoson was shown to have an excellent safety profile in phase II studies and is a phase III study is ongoing. The mechanism of action of namodenoson involves de-regulation of the Wnt and NF-κB signaling pathways, followed by an increase in pro-apoptotic proteins and Fas-ligand, resulting in tumor growth inhibition. The objectives of the currents study were to examine the anti-growth effect of namodenoson on pancreatic carcinoma cells both in vitro and in vivo and to investigate the molecular mechanisms involved. Methods: BxPC-3 human pancreatic carcinoma cells were cultured in the presence and absence of 0.01, 0.1, and 1 nM namodenoson, or in the presence of a combination of namodenoson (0.1 nM) and gemcitabine (0.2 µM). Cell growth was monitored via 3[H]-thymidine proliferation, MTT, and PrestoBlue assays. Western blot analyses were conducted to identify the regulatory cell growth proteins impacted by the treatment. A xenograft model with human BxPC-3 cells in nude mice was established to investigate the in-vivo effect of namodenoson. Results: A significant dose-dependent growth inhibition of BxPC-3 cells was observed with namodenoson in the3[H]-thymidine proliferation assay (1 nM: 67.4% ± 1.7%, p < 0.001; 0.1 nM: 53.7% ± 6.3%, p< 0.05; 0.01 nM: 27.9 % ± 2.3%, p< 0.005). The combination of namodenoson and gemcitabine showed an additive inhibitory effect in the MTT assay (namodenoson: 48.6% ± 1.4%; gemcitabine: 44.4% ± 0.7%; namodenoson plus gemcitabine: 65.4% ± 1.4%; p<0.001 for all). Western blot analyses demonstrated that namodenoson treatment was associated with de-regulation of the Wnt pathway regulatory proteins including p-Akt, NF- κB, GSK-3β, β-catenin and up-regulation of the BAD and BAX apoptotic proteins. In the xenograft model, daily oral treatment with 10 µg/kg of namodenoson led to a significant progressive inhibition of tumor growth. Conclusions: Nanomolar concentrations of namodenoson inhibited the growth of pancreatic carcinoma in cell culture and in a xenograft model. This effect involved A3AR activation and de-regulation of the Wnt/β-catenin pathway. Our results support a potential clinical utility for namodenoson in pancreatic cancer, a disease where an unmet clinical need exists, and novel therapeutic opportunities are required. Citation Format: Pnina Fishman, Avital Bareket-Samish, Inbal Izhak. Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A074.

Multi-Functional Small Molecule Alleviates Fracture Pain and Promotes Bone Healing.

Bone injuries such as fractures are one major cause of morbidities worldwide. A considerable number of fractures suffer from delayed healing, and the unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids with substantial adverse effects. Herein, we tested the hypothesis that the purine molecule, adenosine, can simultaneously alleviate pain and promote healing in a mouse model of tibial fracture by targeting distinctive adenosine receptor subtypes in different cell populations. To achieve this, a biomaterial-assisted delivery of adenosine is utilized to localize and prolong its therapeutic effect at the injury site. The results demonstrate that local delivery of adenosine inhibited the nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigated pain as demonstrated by weight bearing and open field movement tests. Concurrently, local delivery of adenosine at the fracture site promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) resulting in improved bone healing as shown by histological analyses and microCT imaging. This study demonstrates the dual role of adenosine and its material-assisted local delivery as a feasible therapeutic approach to treat bone trauma and associated pain.

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.

Dynamic allosteric networks drive adenosine A1 receptor activation and G-protein coupling.

G-protein coupled receptors (GPCRs) present specific activation pathways and signaling among receptor subtypes. Hence, an extensive knowledge of the structural dynamics of the receptor is critical for the development of therapeutics. Here, we target the adenosine A1 receptor (A1R), for which a negligible number of drugs have been approved. We combine molecular dynamics simulations, enhanced sampling techniques, network theory and pocket detection to decipher the activation pathway of A1R, decode the allosteric networks and identify transient pockets. The A1R activation pathway reveal hidden intermediate and pre-active states together with the inactive and fully-active states observed experimentally. The protein energy networks computed throughout these conformational states successfully unravel the extra and intracellular allosteric centers and the communication pathways that couples them. We observe that the allosteric networks are dynamic, being increased along activation and fine-tuned in presence of the trimeric G-proteins. Overlap of transient pockets and energy networks uncover how the allosteric coupling between pockets and distinct functional regions of the receptor is altered along activation. By an in-depth analysis of the bridge between activation pathway, energy networks and transient pockets, we provide a further understanding of A1R. This information can be useful to ease the design of allosteric modulators for A1R.

Dynamic allosteric networks drive adenosine A1 receptor activation and G-protein coupling

G-protein coupled receptors (GPCRs) present specific activation pathways and signaling among receptor subtypes. Hence, an extensive knowledge of the structural dynamics of the receptor is critical for the development of therapeutics. Here, we target the adenosine A1 receptor (A1R), for which a negligible number of drugs have been approved. We combine molecular dynamics simulations, enhanced sampling techniques, network theory, and pocket detection to decipher the activation pathway of A1R, decode the allosteric networks, and identify transient pockets. The A1R activation pathway reveals hidden intermediate and pre-active states together with the inactive and fully-active states observed experimentally. The protein energy networks computed throughout these conformational states successfully unravel the extra and intracellular allosteric centers and the communication pathways that couple them. We observe that the allosteric networks are dynamic, being increased along activation and fine-tuned in the presence of the trimeric G-proteins. Overlap of transient pockets and energy networks uncovers how the allosteric coupling between pockets and distinct functional regions of the receptor is altered along activation. Through an in-depth analysis of the bridge between the activation pathway, energy networks, and transient pockets, we provide a further understanding of A1R. This information can be useful to ease the design of allosteric modulators for A1R.

Relief of Morphine Withdrawal Symptoms by Japanese Sake Yeast Supplement (Saccharomyces cerevisiae sake) Through Adenosine A1 Receptor Activation in Mice

Background: Morphine withdrawal syndrome is often treated with chemical drugs; however, these drugs can have concerning side effects. Some natural substances might offer safer alternatives for managing withdrawal symptoms. The neuroprotective activity of the adenosine A1 receptor (A1R) in the central nervous system (CNS) has been mentioned before. As a novel natural agent, Japanese sake yeast is enriched with adenosine analogs. Objectives: As the first report, the present study was designed to evaluate the effects of the Japanese sake yeast supplement against the excitatory signs of morphine withdrawal syndrome in mice. Methods: Mice were treated orally (gavage) with sake yeast at doses of 100, 200, and 300 mg kg-1 once daily for a week. Furthermore, during the last 3 days of receiving sake yeast, the animals were made physically dependent on morphine by being given twice-daily subcutaneous injections of increasing doses of morphine (30 - 90 mg/kg) for 3 consecutive days. The withdrawal syndrome was induced in animals through the intraperitoneal (i.p.) administration of naloxone (3 mg kg-1) or was elicited spontaneously. After the examination of withdrawal signs, the animals were decapitated, and their brains were prepared for histopathology. Results: Sake yeast alleviated the intensity of ptosis, piloerection, diarrhea, irritability, and tremor during the two withdrawal syndrome protocols (P

Abstract P2142: Evaluation Of Novel A3 Adenosine Receptor Agonist In Hypertension

The search for alternative strategies for treatment of cardiovascular diseases, lead to the design and synthesis of N -acylhydrazone derivative, named LASSBio-2062, which is a new ligand of adenosine receptor and that is a possible therapeutic target to reduce blood pressure because of its vasodilatory action and antioxidant properties. This work investigated the effects of a new A 3 adenosine receptor agonist (LASSBio-2062) in spontaneously hypertensive rats (SHR). Animal Care and Use Committee at Universidade Federal do Rio de Janeiro approved the protocols (017/19). Vascular reactivity was evaluated using isometric tension recording of pre-contracted thoracic aorta from male Wistar rats after exposure to increasing concentrations of LASSBio-2062 (0.1 - 100 μM) in the absence or presence of antagonist of adenosine A 2A receptor, ZM-241385 (0.1 μM), an antagonist of adenosine A 3 receptor, MRE 3008F20 (0.1 μM), a blocker of K ATP channel, glibenclamide (10 μM), an inhibitor of Kv channels, 4-AP, inhibitor of BK Ca , TEA (3 mM). Mechanical removal of endothelium increased the EC 50 for vasodilation from 15.5 ± 6.5 to 49.4 ± 10.0 μM, indicating partial dependence on the functional integrity of vascular endothelium. Vascular relaxation was not altered by ZM-241385 but the concentration-response curve shifted to the right with MRE 3008F20 and reduced maximum relaxation from 72.1 ± 4.0 to 23.6 ± 7.7%, indicating that vasodilation occurred by activation of the adenosine receptor type A 3 , but not A 2A . Reduced maximum vascular response by glibenclamide, TEA and 4-AP suggests that activation of A 3 adenosine receptor promotes hyperpolarization as a result reduces intracellular calcium. Antihypertensive effect was investigated in spontaneously hypertensive rats (SHR, 12-15 wks old), through the measurement of mean blood pressure (MBP) after intravenous injection of 10 and 30 μmol/kg of LASSBio-2062. After intravenous administration of LASSBio-2062 (10 umol/kg) in SHR, systolic and diastolic pressures reduced from 155.1 ± 10.1 to 110.6 ± 8.0 and from 111.0 ± 9.1 to 63.9 ± 14.7 mmHg, respectively. HR was also changed from 248.1 ± 12.1 to 146.5 ± 21.0 bpm. In conclusion, the N -acylhydrazone, LASSBio-2062 is a potential therapeutic agent to treat arterial hypertension.

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.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.

Effects of namodenoson on pancreatic carcinoma: Preclinical evidence.

e15134 Background: Namodenoson, an A3 adenosine receptor (A3AR) agonist, is currently in a phase 3 trial for the treatment of advanced liver cancer. The mechanism of action of namodenoson involves de-regulation of the Wnt and NF-κB signaling pathways, followed by an increase in pro-apoptotic proteins and Fas-ligand, resulting in tumor growth inhibition. As the Wnt signaling pathway is also highly implicated in pancreatic carcinogenesis, we examined the anti-growth effect of namodenoson on pancreatic carcinoma cell lines and investigated the molecular mechanism involved. Methods: BxPC-3 human pancreatic carcinoma cells were cultured in the presence and absence of 0.01, 0.1, and 1 nM namodenoson. A combined treatment of namodenoson (0.1 nM) and gemcitabine (0.2 µM) was also studied. 3[H]-thymidine proliferation and MTT assays were used to monitor cell growth, and Western blot analyses were performed to identify the involved regulatory cell growth proteins. Results: Results of the 3[H]-thymidine proliferation assays demonstrated significant dose-dependent inhibition of the growth of BxPC-3 cells with namodenoson (1 nM: 67.4% ± 1.7%, p < 0.001; 0.1 nM: 53.7% ± 6.3%, p < 0.05; 0.01 nM: 27.9 % ± 2.3%, p < 0.005). The MTT results revealed that a combined treatment with namodenoson plus gemcitabine had an additive inhibitory effect (namodenoson: 48.6% ± 1.4%; gemcitabine: 44.4% ± 0.7%; namodenoson plus gemcitabine: 65.4% ± 1.4%; p < 0.001 for all). Western blot analyses showed that namodenoson treatment was associated with downregulation of the Wnt pathway regulatory proteins including p-Akt, NF- κB, GSK-3β, and β-catenin. Conclusions: Our findings showed that nanomolar concentrations of namodenoson inhibit the growth of pancreatic carcinoma via A3AR activation and de-regulation of the Wnt/β-catenin pathway, both as a monotherapy and in combination with gemcitabine. Thus, these results support a potential role for namodenoson in treating pancreatic cancer, thereby opening a novel therapeutic opportunity for this disease.

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

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. 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. 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. 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.

A2AR as a Prognostic Marker and a Potential Immunotherapy Target in Human Glioma.

Gliomas are considered one of the most malignant tumors in the body. The immune system has the ability to control the initiation and development of tumors, including gliomas. Thus, immune cells find themselves controlled by various molecular pathways, inhibiting their activation, such as the immunosuppressive adenosine 2A receptor (A2AR). Our objective was to establish the expression profile and role of A2AR at the transcriptomic level, using real-time RT-PCR in Moroccan glioma patients, in addition to TCGA and CGGA cohorts. The real-time RT-PCR results in Moroccan patients showed that high expression of this gene was associated with poor survival in males. Our study on the CGGA cohort corroborated these results. In addition, there was a positive association of A2AR with T-cell exhaustion genes. A2AR also correlated strongly with genes that are primarily enriched in focal adhesion and extracellular matrix interactions, inducing epithelial mesenchymal transition, angiogenesis, and glioma growth. However, in the TCGA cohort, the A2AR showed results that were different from the two previously examined cohorts. In fact, this gene was instead linked to a good prognosis in patients with the astrocytoma histological type. The correlation and enrichment results reinforced the prognostic role of A2AR in this TCGA cohort, in which its high expression was shown to be related to lymphocyte differentiation and a successful cytolytic response, suggesting a more efficient anti-tumor immune response. Correlations and differential analyses based on A2AR gene expression, to understand the cause of the association of this gene with two different prognoses (CGGA males and TCGA Astrocytoma), showed that the overexpression of A2AR in Chinese male patients could be associated with the overexpression of extracellular adenosine, which binds to A2AR to induce immunosuppression and consequently a poor prognosis. However, in the second group (TCGA astrocytomas), the overexpression of the gene could be associated with an adenosine deficiency, and therefore this receptor does not undergo activation. The absence of A2AR activation in these patients may have protected them from immunosuppression, which could reflect the good prognosis. A2AR can be considered a promising therapeutic target in male CGGA and Moroccan patients with gliomas.

Remdesivir-induced conduction abnormalities: A molecular model-based explanation.

Purpose: Remdesivir use in COVID-19 is associated with cardiac conduction abnormalities from unclear mechanisms. A proposed mechanism is the bioaccumulation of the intermediate metabolite GS-441524 resulting in exogenous activation of cardiac adenosine A1 due to the structural similarity between adenosine and GS-441524. The prolonged half-life of GS-441524 can result in sustained activation of adenosine A1 receptors. In this study, we used molecular modeling of adenosine, GS-441524 and the adenosine A1 receptor to assess the potential mechanistic association of the proposed mechanism. Methods: Adenosine and GS-441524 structures were acquired from the PubChem database. Ligand docking was carried out using UCSF Chimera. Models were chosen based on greatest binding affinity and minimum root mean square deviation. Figures of resulting structural models were prepared using UCSF Chimera or PyMOL 2.3.5. Results: By modeling the interaction between the A1 G protein complex and both adenosine and GS-441524, we found that the proposed mechanism of exogenous A1 receptor activation is feasible based on docking compatibility. Conclusion: The proposed mechanism of exogenous cardiac A1 receptor activation from bioaccumulation of GS-441524 as a cause of observed cardiac conduction abnormalities with the use of remdesivir in COVID-19 is viable. Further studies are needed to assess causality.

Dynamic allosteric networks drive adenosine A1 receptor activation and G-protein coupling.

Biological receptors respond to environmental changes through allosteric regulation. In G-protein coupled receptors (GPCRs), the binding of ligands establishes chemical and dynamic communication networks resulting in key structural changes of a transmembrane helix that promotes the coupling and activation of G-proteins. The activation pathways and allosteric mechanisms of GPCRs present system-specific profiles among subtypes. Hence, the development of novel allosteric drugs clearly benefits from a complete characterization of the structural dynamics of the target receptor. In this work, we focus on the adenosine A1 receptor (A1R), for which regardless the progress made in understanding its allosteric modulation though experimental and computational studies, essential knowledge of the allosteric mechanism and communication networks that activates the receptor remains unknown. In particular, we have developed a computational strategy that combines molecular dynamics simulations, enhanced sampling techniques, network theory and pocket detection in order to decipher the interplay between the activation pathway of A1R, the allosteric networks and the transient pockets. Our simulations reveal hidden intermediate and pre-active states together with the inactive and fully-active states observed experimentally. The protein energy networks computed throughout these conformational states successfully unravel the extra and intracellular allosteric centers together with the communication pathways that couples them. We observe that the allosteric networks are dynamic, being increased along activation and fine-tuned in presence of the trimeric G-proteins. Overlap of the transient pockets detected and energy networks uncover how the allosteric coupling between pockets and distinct functional regions of the receptor is altered along activation. This complete dynamic picture provides essential information to ease the design of efficient allosteric modulators for A1R. Our computational workflow can be also applied to other GPCRs and related receptors.

Gene regulation in activated microglia by adenosine A3 receptor agonists: a transcriptomics study.

Most neurodegenerative disorders, including the two most common, Alzheimer's disease (AD) and Parkinson's disease (AD), course with activation of microglia, the resident innate immune cells of thecentral nervous system. A3 adenosine receptor (A3R) agonists have been proposed to be neuroprotective by regulating the phenotype of activated microglia. RNAseq was performed using samples isolated from lipopolysaccharide/interferon-γ activated microglia treated with 2-Cl-IB-MECA, a selective A3R agonist. The results showed that the number of negatively regulated genes in the presence of 2-Cl-IB-MECA was greater than the number of positively regulated genes. Gene ontology enrichment analysis showed regulation of genes participating in several cell processes, including those involved in immune-related events. Analysis of known and predicted protein-protein interactions showed that Smad3 and Sp1 are transcription factors whose genes are regulated by A3R activation. Under the conditions of cell activation and agonist treatment regimen, 2-Cl-IB-MECA did not lead to any tendency to favor the expression of genes related to neuroprotective microglia (M2).