Activation Of Adenosine A1 Receptors Research Articles

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 the central 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).

Repeated topical paeoniflorin attenuates postoperative pain and accelerates cutaneous fibroblast proliferation in mice

This study investigated whether the repeated topical paeoniflorin inhibits postoperative pain in mice. An incision of the plantar skin and underlying muscle of the hind paw elicits acute postoperative pain. Repeated topical paeoniflorin inhibited postoperative pain. An adenosine A1 receptor antagonist (DPCPX) attenuated the analgesic effect of paeoniflorin. Paeoniflorin treatment accelerated wound healing at the surgical site. Paeoniflorin accelerated fibroblast proliferation, which inhibited by DPCPX. These results suggest that the repeated topical paeoniflorin attenuates postoperative pain and accelerated wound healing through fibroblast proliferation, and the activation of adenosine A1 receptor is involved in the action of paeoniflorin.

The Agonist of Adenosine A1 Receptor Induced Desensitization of delta Opioid receptor-mediated Raf-1/MEK/ERK Signaling by Feedback Phosphorylation of Raf-1-Ser289/296/301.

Our previous study found that activation of adenosine A1 receptor (A1R) induced phosphorylation of delta opioid receptor (DOR) and desensitization of its downstream signaling molecules, cAMP and Akt. To further investigate the effect of A1R agonist on DOR signaling and the underlying mechanism, we examined the effect of A1R activation upon binding of its agonist N6-cyclohexyl-adenosine (CHA) on DOR-mediated Raf-1/MEK/ERK activation, and found that prolonged CHA exposure resulted in downregulation of DOR-mediated Raf-1/MEK/ERK signaling pathway. CHA-treatment time dependently attenuated Raf-1-Ser338 phosphorylation induced by [D-Pen2,5] enkephalin (DPDPE), a specific agonist of DOR, and further caused downregulation of the Raf-1/MEK/ERK signaling pathway activated by DOR agonist. Moreover, CHA exposure time-dependently induced the phosphorylation of Raf-1-Ser289/296/301, the inhibitory phosphorylation sites that were regulated by negative feedback, thereby inhibiting activation of the MEK/ERK pathway, and this effect could be blocked by MEK inhibitor U0126. Finally, we proved that the heterologous desensitization of the Raf-1/MEK/ERK cascade was essential in the regulation of anti-nociceptive effect of DOR agonists by confirming that such effect was inhibited by pretreatment of CHA. Therefore, we conclude that the activation of A1R inhibits DOR-mediated MAPK signaling pathway via heterologous desensitization of the Raf-1/MEK/ERK cascade, which is a result of ERK-mediated Raf-1-Ser289/296/301 phosphorylation mediated by activation of A1R.

The impact of Müller cells on development and resolution of macular edema

AbstractMüller cells support physiological neuronal activity and the integrity of the blood‐retinal barrier. A major function of Müller cells is the fluid absorption from the retinal tissue, which is mediated by transcellular water transport coupled to currents through potassium channels. In various disorders as retinal ischemia, ocular inflammation and /or retinal detachment, it was shown that Müller cells decrease the expression of the potassium channel (Kir4.1) which leads to an impairment of the water transport across Müller cell membranes, and induces the cellular swelling. Osmotic swelling of Müller cells is also induced by oxidative stress and by inflammatory mediators. The data suggest that a disturbed fluid transport through Müller cells is (in addition to vascular leakage) a pathogenic factor contributing to the development of retinal edema. Pharmacological re‐activation of the retinal water clearance by Müller cells may be used for treatment of macular edema. Intravitreal steroids as triamcinolone acetonide prevent osmotic swelling of Müller cells and induce the release of endogenous adenosine and subsequent A1 receptor activation which results in the opening of ion channels. Apparently, steroids resolve the edema by both inhibition of vascular leakage and stimulation of retinal fluid clearance by Müller cells.

Purinergic Signalling in the Cochlea

The mammalian cochlea is the sensory organ of hearing with a delicate, highly organised structure that supports unique operating mechanisms. ATP release from the secretory tissues of the cochlear lateral wall (stria vascularis) triggers numerous physiological responses by activating P2 receptors in sensory, supporting and neural tissues. Two families of P2 receptors, ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors, activate intracellular signalling pathways that regulate cochlear development, homeostasis, sensory transduction, auditory neurotransmission and response to stress. Of particular interest is a purinergic hearing adaptation, which reflects the critical role of the P2X2 receptor in adaptive cochlear response to elevated sound levels. Other P2 receptors are involved in the maturation of neural processes and frequency selectivity refinement in the developing cochlea. Extracellular ATP signalling is regulated by a family of surface-located enzymes collectively known as "ectonucleotidases" that hydrolyse ATP to adenosine. Adenosine is a constitutive cell metabolite with an established role in tissue protection and regeneration. The differential activation of A1 and A2A adenosine receptors defines the cochlear response to injury caused by oxidative stress, inflammation, and activation of apoptotic pathways. A1 receptor agonism, A2A receptor antagonism, and increasing adenosine levels in cochlear fluids all represent promising therapeutic tools for cochlear rescue from injury and prevention of hearing loss.

Selective reduction of neurotransmitter release by cAMP-dependent pathways in mouse detrusor.

Parasympathetic nerve-mediated contractions of detrusor smooth muscle are generated by ATP and acetylcholine (ACh) release from efferent nerve terminals. In humans, ACh is responsible for detrusor contractions in normal human bladders, whereas ATP has an additional role in overactive bladder pathologies. The ATP metabolite, adenosine, relaxes nerve-mediated contractions, with a potential action via presynaptic adenosine A1 receptor activation and subsequent suppression of neuronal ATP release. We investigated the effect of A1 receptor activation and downstream cAMP-dependent pathways on nerve-mediated ATP and ACh release, and detrusor contraction in mouse detrusor. Bladders from male C57BL/6 mice (12 wk) were used for in vitro experiments. Upon electrical field stimulation of intact preparations (detrusor and mucosal layers), ATP or ACh release was measured simultaneously with tension recordings. Activation of A1 receptors by adenosine or exogenous agonists reduced the lower frequency component of nerve-mediated contractions and neuronal ATP release. The A1 receptor antagonist abolished these effects. A1 receptor activation inhibits adenylyl cyclase (AC) activity and cAMP generation. The effect of A1 receptor activation was mimicked by a PKA antagonist but not by modulators of exchange proteins activated by cAMP, demonstrating that modulation of nerve-mediated ATP release is via PKA. Adenosine had no effect on ACh release or the higher frequency component of nerve-mediated contractions. Differential regulation of neurotransmitter release is possible at the detrusor nerve-muscle junction, as demonstrated by A1 receptor activation, and downstream inhibition of AC, cAMP generation, and PKA. The ability to specifically attenuate ATP release offers a potential to target purinergic motor pathways enhanced in overactive bladder pathologies.

Adenosine A1 receptor antagonist-induced facilitation of postsynaptic AMPA currents in pyramidal neurons of the rat hippocampal CA2 area.

Adenosine A1 receptors (A1R) are widely expressed in hippocampal pyramidal neurons and their presynaptic terminals. It is well known that endogenous adenosine regulates hippocampal function through the activation of A1R in hippocampal pyramidal neurons and has been reported that blockade of A1R induces stronger potentiation of excitatory synaptic transmission in CA2 pyramidal neurons than in CA1 pyramidal neurons. This strong potentiation of CA2 neurons is thought to be caused by the specific modulation of excitatory synaptic transmission through postsynaptic A1R. However, the direct effects of A1R on postsynaptic AMPA channels remain unknown because of the technical difficulties of patch-clamp recording from mature hippocampal CA2 neurons. We recorded synaptic currents from pyramidal neurons in CA1 and CA2 and analyzed the effects of an A1R antagonist on stimulation-evoked synaptic transmission and local application-induced postsynaptic AMPA currents. The antagonist increased the amplitude of evoked synaptic transmission in neurons in both CA1 and CA2. This facilitation was larger in pyramidal neurons in CA2 than in CA1. The antagonist also increased postsynaptic AMPA currents in neurons in CA2 but not in CA1. This facilitation of CA2 AMPA currents was occluded by the intracellular application of a G-protein blocker. Even with the blockade of postsynaptic G-protein signaling, the A1R antagonist increased evoked synaptic transmission in neurons in CA2. These results suggest that synaptic transmission in pyramidal neurons in CA2 is regulated by both presynaptic and postsynaptic A1R. Moreover, A1R regulate excitatory synaptic transmission in pyramidal neurons in CA2 through the characteristic postsynaptic modulation of AMPA currents.

A review for the pharmacological effects of paeoniflorin in the nervous system.

Paeoniflorin, a terpenoid glycoside compound extracted from Paeonia lactiflora Pall, shows preventive and therapeutic effects in various types of nervous system disorders. However, to date, no comprehensive knowledge on the pharmacological effects of paeoniflorin on the nervous system is available online. Clarification of this issue may be useful for the development of paeoniflorin as a new drug for the treatment of nervous system disorders. To this end, the authors summarize the pharmacological aspects of paeoniflorin and its possible mechanisms, such as restoration of mitochondrial function; inhibition of neuroinflammation, oxidative stress, and cellular apoptosis; activation of adenosine A1 receptor, cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase 1/2 (ERK1/2); or enhancement of brain-derived neurotrophic factor and serotonin function, in the prevention of disorders such as cerebral ischemia, subarachnoid hemorrhage, vascular dementia, Alzheimer’s disease, Parkinson’s disease, depression, post-traumatic syndrome disorder, and epilepsy, by reviewing the previously published literature.

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.

Modulation of adenosine signaling reverses 3-nitropropionic acid-induced bradykinesia and memory impairment in adult zebrafish

Huntington's disease (HD) is a neurodegenerative disorder, characterized by motor dysfunction, psychiatric disturbance, and cognitive decline. In the early stage of HD, occurs a decrease in dopamine D2 receptors and adenosine A2A receptors (A2AR), while in the late stage also occurs a decrease in dopamine D1 receptors and adenosine A1 receptors (A1R). Adenosine exhibits neuromodulatory and neuroprotective effects in the brain and is involved in motor control and memory function. 3-Nitropropionic acid (3-NPA), a toxin derived from plants and fungi, may reproduce HD behavioral phenotypes and biochemical characteristics. This study investigated the effects of acute exposure to CPA (A1R agonist), CGS 21680 (A2AR agonist), caffeine (non-selective of A1R and A2AR antagonist), ZM 241385 (A2AR antagonist), DPCPX (A1R antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in an HD pharmacological model induced by 3-NPA in adult zebrafish. CPA, CGS 21680, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered via i.p. in zebrafish after 3-NPA (at dose 60 mg/kg) chronic treatment. Caffeine and ZM 241385 reversed the bradykinesia induced by 3-NPA, while CGS 21680 potentiated the bradykinesia caused by 3-NPA. Moreover, CPA, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA reversed the 3-NPA-induced memory impairment. Together, these data support the hypothesis that A2AR antagonists have an essential role in modulating locomotor function, whereas the activation of A1R and blockade of A2AR and A1R and modulation of adenosine levels may reduce the memory impairment, which could be a potential pharmacological strategy against late-stage symptoms HD.

Investigating the temperature‐dependent and independent neuroprotective effects of cyclohexyladenosine in SH‐SY5Y neurons undergoing oxygen glucose deprivation

IntroductionStroke is a global leading cause of adult disability and death. Hypothermia is the most potent neuroprotectant preclinically. It acts broadly upon the pathophysiological mechanisms of ischemia‐induced injury. Clinical trials using physical cooling, which triggers compensatory thermogenesis, have failed. Activation of hypothalamic A1 adenosine receptors (A1AR) by N6‐cyclohexyladenosine (CHA) suppresses thermogenesis in several species. Activation of non‐hypothalamic CNS A1AR at 37 °C also reduces neuronal death. CHA has neuroprotective potential, but the contributions of temperature‐dependent and independent effects are unknown. Therefore, we studied the effects of hypothermia and/or CHA upon the viability of neuronal cultures undergoing oxygen glucose deprivation (OGD). Hypothesis Both temperature‐dependent and independent effects of CHA contribute to neuroprotection in SH‐SY5Y neuronal cultures against OGD.MethodsNeuronal OGD model: SH‐SY5Y human neuroblastoma cells were cultured in Dulbecco modified Eagle's medium with 10% fetal bovine serum, penicillin and streptomycin, and maintained at 37 °C in 95% N2, 5% CO2 in a humidified incubator. Cultures underwent 8 h OGD in a hypoxic chamber at 37 or 33 °C, then 24 h of normoxia in standard culture media, to simulate reperfusion. Drug: N6‐cyclohexyladenosine was dissolved in ethanol to a 1 mM stock and used at 1 μM. Treatment groups: Stable cultures were randomly assigned to: normoxia, 8 h OGD at 37 °C and 8 h OGD and 33 °C. Within each group, wells were assigned to CHA 1 μM, vehicle, or medium only (given 24 h before OGD, during OGD and for 24 h after OGD). One‐way ANOVA was used for statistical comparisons. Outcome measures: Cell death was assessed by flow cytometry (Cytek Aurora 4L). Briefly, neurons were detached using EDTA, washed twice, and resuspended to 106 cells per sample. Cells were incubated in the dark with Zombie Red (Biolegend; 15 min, room temperature, 1:1500 dilution) and then Caspase 3/7 (CellEvent; 30 min, 37 °C,1:500 dilution) and washed twice. Samples (>10,000 events) were gated to exclude debris and for subpopulations of healthy/apoptotic/necrotic cells.Results8 h OGD significantly decreased neuronal viability (37.1 ± 0.7%; mean ± SEM) when compared with normoxic control (70.1 ± 4.0%) (Fig. 1). 33 °C hypothermia was significantly protective (51.9 ± 2.9% healthy cells; p<0.05) of neurons. CHA was significantly protective both at 37 °C (46.7 ± 2.8%; p<0.05) and when combined with hypothermia at 33 °C (69.3 ± 2.6%; p<0.05). There were no significant changes in the percentages of early apoptotic or late apoptotic/necrotic cells.ConclusionCHA provides neuroprotective effect that is additive to that provided by hypothermia in SH‐SY5Y cells. Therefore, it is likely that neuroprotection from CHA in vivo not only can be achieved via its hypothermic effects but also direct temperature‐independent effects on presynaptic cortical neurons.

The mechanism study of YZG-331 on sedative and hypnotic effects

YZG-331 is a synthetic novel derivates of N6-(4-hydroxybenzyl) adenine riboside (NHBA), which has potent sedative and hypnotic effects based on our previous study. We are now aiming to investigate the mechanism of YZG-331. In this research, the behavioral studies showed that YZG-331 (4, 8, 16 mg/kg, i.g.) could reduce the spontaneous locomotor activity in mice, which could be blocked by AM (non-selective adenosine receptor antagonist), DPCPX (adenosine A1 receptor (A1R) antagonist), and SCH58261 (adenosine A2a receptor (A2aR) antagonist). Moreover, YZG-331 no longer exerted sedative effect in A1R or A2aR knockdown mice. YZG-331 (2.5, 5, 10 mg/kg, i.g.) prolonged sleeping time in pentobarbital sodium treated mice, which can be prevented by DPCPX or SCH58261. The above results demonstrated that YZG-331 exerted sedative and hypnotic effects through A1R and A2aR. In addition, it was found that YZG-331 (25, 50, 100 μM) decreased intracellular calcium level and YZG-331 (10 mg/kg, i.g.) decreased CaMKII phosphorylation (pCaMKII) level in mouse hypothalamus and cortex. In summary, this study indicated that activation of A1R/ A2aR and down regulation of Ca2+-CaMKII signaling pathway were involved in the sedative and hypnotic effects of YZG-331.

Discovery of Pyridone-Substituted Triazolopyrimidine Dual A2A/A1 AR Antagonists for the Treatment of Ischemic Stroke.

Ischemic stroke is a complex systemic disease characterized by high morbidity, disability, and mortality. The activation of the presynaptic adenosine A2A and A1 receptors modifies a variety of brain insults from excitotoxicity to stroke. Therefore, the discovery of dual A2A/A1 adenosine receptor (AR)-targeting therapeutic compounds could be a strategy for the treatment of ischemic stroke. Inspired by two clinical phase III drugs, ASP-5854 (dual A2A/A1 AR antagonist) and preladenant (selective A2A AR antagonist), and using the hybrid medicinal strategy, we characterized novel pyridone-substituted triazolopyrimidine scaffolds as dual A2A/A1 AR antagonists. Among them, compound 1a exerted excellent A2A/A1 AR binding affinity (Ki = 5.58/24.2 nM), an antagonistic effect (IC50 = 5.72/25.9 nM), and good metabolic stability in human liver microsomes, rat liver microsomes, and dog liver microsomes. Importantly, compound 1a demonstrated a dose–effect relationship in the oxygen-glucose deprivation/reperfusion (OGD/R)-treated HT22 cell model. These findings support the development of dual A2A/A1 AR antagonists as a potential treatment for ischemic stroke.

CF101 alleviates OA progression and inhibits the inflammatory process via the AMP/ATP/AMPK/mTOR axis.

CF101 (IB-MECA) is an adenosine A3 receptor agonist that has anti-inflammatory and pain-relieving properties. Adenosine A3 receptor activation can delay the process of Osteoarthritis(OA) and prevent the occurrence of OA. However, the mechanism of CF101 on OA is still unknown. This study aimed to investigate the effect of CF101 on rats induced by anterior cruciate ligament-transection (ACLT) and rat chondrocytes induced by IL-1ß. ACLT-induced OA rats were administered CF101, and autophagy levels were measured to determine whether CF101 had an autophagy-mediated protective effect on articular cartilage. Furthermore, the mechanism by which CF101 protected articular cartilage in IL-1ß-induced chondrocytes mimicking OA was investigated. In rats treated with ACLT, CF101 was able to delay the progression of OA, as well as reduce inflammation and type II collagen degradation factors. In addition, in vitro experiments revealed that CF101 reduced type II collagen degradation factors in OA chondrocytes. In rats treated with ACLT and OA chondrocytes, CF101 enhanced autophagy and increased the ratio of AMP/ATP and AMPK protein levels while decreasing mTOR expression. Treatment of OA chondrocytes with 3-MA prior to treatment with CF101 resulted in inhibition of autophagy factor levels, as well as increased levels of inflammatory factors and type II collagen degradation compared to the CF101 group. These findings demonstrated that CF101 could protect articular cartilage against OA by enhancing the ratio of ATP/AMP and altering the AMPK/mTOR pathway to enhance autophagy and reduce inflammation. In addition, inhibition of autophagy resulted in a reduced CF101 effect.

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.

Mitochondrial respiration in rats during hypothermia resulting from central drug administration.

The ability to induce a hypothermia resembling that of natural torpor would be greatly beneficial in medical and non-medical fields. At present, two procedures based on central nervous pharmacological manipulation have been shown to be effective in bringing core body temperature well below 30°C in the rat, a non-hibernator: the first, based on the inhibition of a key relay in the central thermoregulatory pathway, the other, based on the activation of central adenosine A1 receptors. Although the role of mitochondria in the activation and maintenance of torpor has been extensively studied, no data are available for centrally induced hypothermia in non-hibernators. Thus, in the present work the respiration rate of mitochondria in the liver and in the kidney of rats following the aforementioned hypothermia-inducing treatments was studied. Moreover, to have an internal control, the same parameters were assessed in a well-consolidated model, i.e., mice during fasting-induced torpor. Our results show that state 3 respiration rate, which significantly decreased in the liver of mice, was unchanged in rats. An increase of state 4 respiration rate was observed in both species, although it was not statistically significant in rats under central adenosine stimulation. Also, a significant decrease of the respiratory control ratio was detected in both species. Finally, no effects were detected in kidney mitochondria in both species. Overall, in these hypothermic conditions liver mitochondria of rats remained active and apparently ready to be re-activated to produce energy and warm up the cells. These findings can be interpreted as encouraging in view of the finalization of a translational approach to humans.

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.

Septotemporal variation in modulation of synaptic transmission, paired-pulse ratio and frequency facilitation/depression by adenosine and GABAB receptors in the rat hippocampus.

Short-term synaptic plasticity represents a fundamental mechanism in neural information processing and is regulated by neuromodulators. Here, using field recordings from the CA1 region of adult rat hippocampal slices, we show that excitatory synaptic transmission is suppressed by strong but not moderate activation of adenosine A1 receptors by 2-Chloro-N6-cyclopentyladenosine (CCPA) more in the dorsal than the ventral hippocampus; in contrast, both mild and strong activation of GABAB receptors by baclofen (1 μM, 10 μM) suppress synaptic transmission more in the ventral than the dorsal hippocampus. Using a 10-pulse stimulation train of variable frequency, we found that CCPA modulates short-term synaptic plasticity independently of the suppression of synaptic transmission in both segments of the hippocampus and at stimulation frequencies greater than 10 Hz. However, specifically regarding the paired-pulse ratio (PPR) and frequency facilitation/depression (FF/D) we found significant drug action before but not after adjusting conditioning responses to control levels. Activation of GABABRs by baclofen suppressed synaptic transmission more in the ventral than the dorsal hippocampus. Furthermore, relatively high (10 μM) but not low (1 μM) baclofen concentration enhanced both PPR and FF in both hippocampal segments at stimulation frequencies greater than 1 Hz, independently of the suppression of synaptic transmission by baclofen. These results show that A1Rs and GABABRs control synaptic transmission more effectively in the dorsal and the ventral hippocampus, respectively, and suggest that these receptors modulate PPR and FF/D at different frequency bands of afferent input, in both segments of the hippocampus.