The association of gene polymorphisms of adenosine and dopamine receptors with the response to caffeine citrate treatment in infants with apnea of prematurity: a prospective nested case-control study.

BACKGROUND: To investigate the potential influence of adenosine and dopamine receptor genes polymorphisms in combination with clinical factors on the response of preterm infants to caffeine citrate treatment in apnea of prematurity (AOP). METHODS: A prospective nested case-control study enrolled 221 preterm infants with gestational age < 34 weeks. These infants were divided into the response (n = 160) and the non-response groups (n = 61). 22 single-nucleotide polymorphisms in adenosine and dopamine receptor genes were genotyped. The basic characteristics and clinical outcomes of the two groups were compared. Univariate logistic regression analysis was performed to evaluate the differences in genotype distribution between the groups. Multivariable logistic regression analysis was performed to identify independent risk and protective factors and develop a nomogram to predict caffeine citrate response in preterm infants. RESULTS: Preterm infants in the non-response group had lower gestational age, lower birth weight, longer periods of oxygen supplementation and caffeine citrate use, and higher incidence of patent ductus arteriosus (PDA), bronchopulmonary dysplasia (BPD), neonatal respiratory distress syndrome (NRDS), retinopathy of prematurity (ROP), and brain injury (P < 0.05 for all). The ADORA1 rs10920573, ADORA2B rs2015353, ADORA3 rs10776728, DRD3 rs7625282, and DRD3 rs6280 gene polymorphisms were associated with caffeine citrate response in preterm infants (PFDR < 0.05 for all). The ADORA1 rs10920573 CC (aOR, 3.51; 95% CI, 1.34-9.25) and DRD3 rs6280 CT genotypes (aOR, 3.19; 95% CI, 1.53-6.65) were independent risk factors for non-response, whereas greater gestational age (aOR, 0.631; 95% CI, 0.53-0.75) was an independent protective factor for response. The concordance index of the nomogram was 0.764 (95% CI, 0.687-0.842), and the calibration and decision curve analysis indicated the nomogram had excellent predict performance. CONCLUSIONS: Adenosine receptor gene and dopamine receptor gene polymorphisms influence caffeine citrate treatment response in AOP. By combining genetic and clinical variables, it is possible to predict the response to caffeine citrate treatment in preterm infants.

Elevated CD39+T-Regulatory Cells and Reduced Levels of Adenosine Indicate a Role for Tolerogenic Signals in the Progression from Moderate to Severe COVID-19.

Viral infections trigger inflammation by controlling ATP release. CD39 ectoenzymes hydrolyze ATP/ADP to AMP, which is converted by CD73 into anti-inflammatory adenosine (ADO). ADO is an anti-inflammatory and immunosuppressant molecule which can enhance viral persistence and severity. The CD39-CD73-adenosine axis contributes to the immunosuppressive T-reg microenvironment and may affect COVID-19 disease progression. Here, we investigated the link between CD39 expression, mostly on T-regs, and levels of CD73, adenosine, and adenosine receptors with COVID-19 severity and progression. Our study included 73 hospitalized COVID-19 patients, of which 33 were moderately affected and 40 suffered from severe infection. A flow cytometric analysis was used to analyze the frequency of T-regulatory cells (T-regs), CD39+ T-regs, and CD39+CD4+ T-cells. Plasma concentrations of adenosine, IL-10, and TGF-ß were quantified via an ELISA. An RT-qPCR was used to analyze the gene expression of CD73 and adenosine receptors (A1, A2A, A2B, and A3). T-reg cells were higher in COVID-19 patients compared to healthy controls (7.4 ± 0.79 vs. 2.4 ± 0.28; p < 0.0001). Patients also had a higher frequency of the CD39+ T-reg subset. In addition, patients who suffered from a severe form of the disease had higher CD39+ T-regs compared with moderately infected patients. CD39+CD4+ T cells were increased in patients compared to the control group. An analysis of serum adenosine levels showed a marked decrease in their levels in patients, particularly those suffering from severe illness. However, this was paralleled with a marked decline in the expression levels of CD73. IL-10 and TGF-ß levels were higher in COVID-19; in addition, their values were also higher in the severe group. In conclusion, there are distinct immunological alterations in CD39+ lymphocyte subsets and a dysregulation in the adenosine signaling pathway in COVID-19 patients which may contribute to immune dysfunction and disease progression. Understanding these immunological alterations in the different immune cell subsets and adenosine signaling provides valuable insights into the pathogenesis of the disease and may contribute to the development of novel therapeutic approaches targeting specific immune mechanisms.

Functioning and Gene Expression of Adenosine A1 Receptor During Zebrafish (Danio rerio) Development.

The A1 adenosine receptor is the most widely expressed P1 receptor in vertebrates, performing inhibitory tone of the nervous system. Increased levels of adenosine are crucial to promote tissue protection in threatening situations, such as convulsion and hypoxia. Zebrafish is an established model organism for studies on health and disease. In this study, we evaluated the functionality of A1 adenosine receptor through development of zebrafish (6-7-day-, 3-, 8-, and 24-month-old), assessing: (I) the effects of the agonist N6-cyclopenthyladenosine (CPA) over locomotor parameters, (II) the anticonvulsant properties of CPA and adenosine per se in the pentylenetetrazol-induced seizure, and (III) the gene expression of adora1b through development. CPA promoted decreased distance traveled in the highest concentrations/doses tested (larvae: 75 to 500 µM; adults: 20 mg.kg-1), altered mean velocity (larvae: 50-500 µM; adults: 20 mg.kg-1) and time in the bottom zone of apparatus (adults: decrease in 20 mg.kg-1). Adenosine increased the latency of the larvae to reach stage II at 5 and 10 µM. CPA anticonvulsant effect against convulsive stage II was reached at 75 µM, although it decreased basal locomotor activity in larvae. For adults, CPA 10 mg.kg-1 was effective as anticonvulsant without locomotory effects. Adenosine had minor anticonvulsant effects in the concentration tested (larvae: 5 and 10 µM). The level of gene expression of adora1b was stable in brain from adult animals (8- and 24-month-old animals). These results suggest that zebrafish has similar responses to CPA as mammals. To avoid confounding factors, such as locomotor effects, during any brain function investigation using A1 adenosine receptor as a target, the concentration below 75 µM or below the dose of 20 mg.kg-1 of CPA is ideal for zebrafish at larval and adult stages, respectively.

Acute caffeine differently affects risk-taking and the expression of BDNF and of adenosine and opioid receptors in rats with high or low anxiety-like behavior.

Anxiety disorders are common psychiatric conditions with a partially elucidated neurobiology. Caffeine, an unspecific adenosine receptor antagonist, is a common psychostimulant with anxiogenic effects in sensitive individuals. High doses of caffeine produce anxiety-like behavior in rats but it is not known if this is specific for rats with high baseline anxiety-like behavior. Thus, the aim of this study was to investigate general behavior, risk-taking, and anxiety-like behavior, as well as mRNA expression (adenosine A2A and A1, dopamine D2, and, µ, κ, δ opioid, receptors, BDNF, c-fos, IGF-1) in amygdala, caudate putamen, frontal cortex, hippocampus, hypothalamus, after an acute dose of caffeine. Untreated rats were screened using the elevated plus maze (EPM), giving each rat a score on anxiety-like behavior based on their time spent in the open arms, and categorized into a high or low anxiety-like behavior group accordingly. Three weeks after categorization, the rats were treated with 50 mg/kg caffeine and their behavior profile was studied in the multivariate concentric square field (MCSF) test, and one week later in the EPM. qPCR was performed on selected genes and corticosterone plasma levels were measured using ELISA. The results demonstrated that the high anxiety-like behavior rats treated with caffeine spent less time in risk areas of the MCSF and resituated towards the sheltered areas, a behavior accompanied by lower mRNA expression of adenosine A2A receptors in caudate putamen and increased BDNF expression in hippocampus. These results support the hypothesis that caffeine affects individuals differently depending on their baseline anxiety-like behavior, possibly involving adenosine receptors. This highlights the importance of adenosine receptors as a possible drug target for anxiety disorders, although further research is needed to fully elucidate the neurobiological mechanisms of caffeine on anxiety disorders.

Gene expression pattern of adenosine receptors in lung tumors.

BACKGROUND: Adenosine, a purine nucleoside, plays an important function in the pathogenesis of cancer through interaction with the cell surface G protein-coupled adenosine receptors. It is important to determine the expression pattern of these receptors in different cancers. Previously in our lab, we found up-regulation of A1 adenosine receptor (AR) in lung tumors playing as a putative target for cancer cell inhibition, and here we aimed to investigate the significance of other adenosine receptor isoforms (A2aAR, A2bAR, and A3AR). METHODS: In this study, first of all, we evaluated the adenosine receptors gene expression in the bioinformatics database (GENT2). Then the genes expression was measured experimentally in the 20 lung cancer tumor tissues in comparison to the matched tumor-adjacent normal tissue (as control). The mRNA expression of receptors was evaluated by real-time PCR. The tumors were categorized by the tumor size and the gene expression change was evaluated. RESULTS: The experimental results indicated a significant increase in A2aAR (p value = .021) and A3AR (p value = .01) expression in lung tumor tissues compared to the adjacent tumor margins which were in accordant to bioinformatics analysis. We found a non-significant increase in A2bAR expression; however, when comparing the patients according to the tumor size, our data showed that the expression of A2bAR adenosine receptor in patients with smaller lung tumor sizes was higher than the other group (p = .011). CONCLUSION: The results of this study showed that adenosine receptors A3AR, and A2aAR are highly expressed in lung tumors relative to tumor-adjacent normal tissue. We suggest that overexpression of adenosine receptors in lung cancer is due to their regulatory role in various aspects of lung cancer.

Changes in adenosine receptors and neurotrophic factors in the SOD1G93A mouse model of amyotrophic lateral sclerosis: Modulation by chronic caffeine.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of corticospinal tract motor neurons. Previous studies showed that adenosine-mediated neuromodulation is disturbed in ALS and that vascular endothelial growth factor (VEGF) has a neuroprotective function in ALS mouse models. We evaluated how adenosine (A1R and A2AR) and VEGF (VEGFA, VEGFB, VEGFR-1 and VEGFR-2) system markers are altered in the cortex and spinal cord of pre-symptomatic and symptomatic SOD1G93A mice. We then assessed if/how chronic treatment of SOD1G93A mice with a widely consumed adenosine receptor antagonist, caffeine, modulates VEGF system and/or the levels of Brain-derived Neurotrophic Factor (BDNF), known to be under control of A2AR. We found out decreases in A1R and increases in A2AR levels even before disease onset. Concerning the VEGF system, we detected increases of VEGFB and VEGFR-2 levels in the spinal cord at pre-symptomatic stage, which reverses at the symptomatic stage, and decreases of VEGFA levels in the cortex, in very late disease states. Chronic treatment with caffeine rescued cortical A1R levels in SOD1G93A mice, bringing them to control levels, while rendering VEGF signaling nearly unaffected. In contrast, BDNF levels were significantly affected in SOD1G93A mice treated with caffeine, being decreased in the cortex and increased in spinal the cord. Altogether, these findings suggest an early dysfunction of the adenosinergic system in ALS and highlights the possibility that the negative influence of caffeine previously reported in ALS animal models results from interference with BDNF rather than with the VEGF signaling molecules.

Crosstalk between adenosine receptors and CYP450-derived oxylipins in the modulation of cardiovascular, including coronary reactive hyperemic response.

Adenosine is a ubiquitous endogenous nucleoside or autacoid that affects the cardiovascular system through the activation of four G-protein coupled receptors: adenosine A1 receptor (A1AR), adenosine A2A receptor (A2AAR), adenosine A2B receptor (A2BAR), and adenosine A3 receptor (A3AR). With the rapid generation of this nucleoside from cellular metabolism and the widespread distribution of its four G-protein coupled receptors in almost all organs and tissues of the body, this autacoid induces multiple physiological as well as pathological effects, not only regulating the cardiovascular system but also the central nervous system, peripheral vascular system, and immune system. Mounting evidence shows the role of CYP450-enzymes in cardiovascular physiology and pathology, and the genetic polymorphisms in CYP450s can increase susceptibility to cardiovascular diseases (CVDs). One of the most important physiological roles of CYP450-epoxygenases (CYP450-2C & CYP2J2) is the metabolism of arachidonic acid (AA) and linoleic acid (LA) into epoxyeicosatrienoic acids (EETs) and epoxyoctadecaenoic acid (EpOMEs) which generally involve in vasodilation. Like an increase in coronary reactive hyperemia (CRH), an increase in anti-inflammation, and cardioprotective effects. Moreover, the genetic polymorphisms in CYP450-epoxygenases will change the beneficial cardiovascular effects of metabolites or oxylipins into detrimental effects. The soluble epoxide hydrolase (sEH) is another crucial enzyme ubiquitously expressed in all living organisms and almost all organs and tissues. However, in contrast to CYP450-epoxygenases, sEH converts EETs into dihydroxyeicosatrienoic acid (DHETs), EpOMEs into dihydroxyoctadecaenoic acid (DiHOMEs), and others and reverses the beneficial effects of epoxy-fatty acids leading to vasoconstriction, reducing CRH, increase in pro-inflammation, increase in pro-thrombotic and become less cardioprotective. Therefore, polymorphisms in the sEH gene (Ephx2) cause the enzyme to become overactive, making it more vulnerable to CVDs, including hypertension. Besides the sEH, ω-hydroxylases (CYP450-4A11 & CYP450-4F2) derived metabolites from AA, ω terminal-hydroxyeicosatetraenoic acids (19-, 20-HETE), lipoxygenase-derived mid-chain hydroxyeicosatetraenoic acids (5-, 11-, 12-, 15-HETEs), and the cyclooxygenase-derived prostanoids (prostaglandins: PGD2, PGF2α; thromboxane: Txs, oxylipins) are involved in vasoconstriction, hypertension, reduction in CRH, pro-inflammation and cardiac toxicity. Interestingly, the interactions of adenosine receptors (A2AAR, A1AR) with CYP450-epoxygenases, ω-hydroxylases, sEH, and their derived metabolites or oxygenated polyunsaturated fatty acids (PUFAs or oxylipins) is shown in the regulation of the cardiovascular functions. In addition, much evidence demonstrates polymorphisms in CYP450-epoxygenases, ω-hydroxylases, and sEH genes (Ephx2) and adenosine receptor genes (ADORA1 & ADORA2) in the human population with the susceptibility to CVDs, including hypertension. CVDs are the number one cause of death globally, coronary artery disease (CAD) was the leading cause of death in the US in 2019, and hypertension is one of the most potent causes of CVDs. This review summarizes the articles related to the crosstalk between adenosine receptors and CYP450-derived oxylipins in vascular, including the CRH response in regular salt-diet fed and high salt-diet fed mice with the correlation of heart perfusate/plasma oxylipins. By using A2AAR-/-, A1AR-/-, eNOS-/-, sEH-/- or Ephx2-/-, vascular sEH-overexpressed (Tie2-sEH Tr), vascular CYP2J2-overexpressed (Tie2-CYP2J2 Tr), and wild-type (WT) mice. This review article also summarizes the role of pro-and anti-inflammatory oxylipins in cardiovascular function/dysfunction in mice and humans. Therefore, more studies are needed better to understand the crosstalk between the adenosine receptors and eicosanoids to develop diagnostic and therapeutic tools by using plasma oxylipins profiles in CVDs, including hypertensive cases in the future.

AdoR-1 (Adenosine Receptor) Contributes to Protection against Paraquat-Induced Oxidative Stress in Caenorhabditis elegans.

AdoR-1, the single adenosine receptor homolog in Caenorhabditis elegans, which belongs to the superfamily of G-protein coupled receptors (GPCRs), mediates most of the physiological effects of extracellular adenosine. Adenosine has been proved to improve the survival rate of C. elegans in oxidative stress conditions. However, the potential mechanism of adenosine's protective effect against oxidative stress via AdoR-1 has not been studied. In this study, C. elegans were divided into three groups: two groups with paraquat treatment, one in the presence and one in the absence of adenosine, and an untreated control group. Results indicate that many differentially expressed genes were found to be enriched significantly in neural-related signaling pathways among transcriptome data of three groups. Further gene network analysis showed that some important genes well known to be involved in promoting the acetylcholine release pathway, such as dop-1, egl-30, and unc-13, and those involved in promoting the neuropeptide release pathway, such as kin-1, were upregulated by paraquat induction but downregulated after adenosine treatment. Meanwhile, a completely opposite trend was observed for the goa-1 gene that inhibits the acetylcholine-release and neuropeptide-release pathway. Additionally, some biochemical assays including SOD, GSSG, GSH, and AChE were measured to identify the potential protection of adenosine against oxidative stress between wild-type strain N2 and ador-1 gene knockout strain EG6890. Conclusively, our study revealed series of adenosine receptor-mediated genes in C. elegans that might act as regulators of paraquat-induced oxidative stress and may indicate adenosine's promising protective effects.

An Ancient Adenosine Receptor Gains Olfactory Function in Bony Vertebrates.

Nucleotides are an important class of odorants for aquatic vertebrates such as frogs and fishes, but also have manifold signaling roles in other cellular processes. Recently, an adenosine receptor believed to belong to the adora2 clade has been identified as an olfactory receptor in zebrafish. Here, we set out to elucidate the evolutionary history of both this gene and its olfactory function. We have performed a thorough phylogenetic study in vertebrates, chordates and their sister group, ambulacraria, and show that the origin of the zebrafish olfactory receptor gene can be traced back to the most recent common ancestor of all three groups as a segregate sister clade (adorb) to the adora gene family. Eel, carp, and clawed frog all express adorb in a sparse and distributed pattern within their olfactory epithelium very similar to the pattern observed for zebrafish that is, consistent with a function as olfactory receptor. In sharp contrast, lamprey adorb-expressing cells are absent from the sensory region of the lamprey nose, but form a contiguous domain directly adjacent to the sensory region. Double-labeling experiments confirmed the expression of lamprey adorb in nonneuronal cells and are consistent with an expression in neuronal progenitor cells. Thus, adorb may have undergone a switch of function in the jawed lineage of vertebrates towards a role as olfactory receptor.

A nonolfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features.

Adenosine receptors (ADORs) are G protein-coupled purinoceptors that have several functions including regulation of chloride secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic and expression analyses indicate that elasmobranch ADORs are nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We coexpressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N6-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A0 core that revealed unique structural features of ancestral receptors. We conclude that 1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A0 likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A0 appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.

Systematic deletion of adenosine receptors reveals novel roles in inflammation and pyroptosis in THP-1 macrophages.

Macrophages perform the fundamental function of sensing cellular damage, initiating and mediating immune response and tissue repair. Adenine nucleotides are in relatively high abundance in cells and are released from cells during tissue damage that are converted to adenosine in the extracellular environment. The A1, A2A, A2B and A3 adenosine receptors serve to regulate immune function. Despite characterization of the adenosine receptors, a comprehensive examination of adenosine receptor signaling in THP-1 macrophage cells has not been done. Moreover, previous studies employed chemical agonists and antagonists that have the potential for off-target affects. Here we systematically knockdown each of the four known adenosine receptors in THP-1 macrophages using validated siRNA and investigated their function under LPS stimulation. We demonstrate that the A1 receptor is required for adenosine-stimulated IL-10 and IL-1ß secretion indicating an important role of this receptor during resolution of inflammation and tissue repair in these cells. The A1 and A3 receptor were required for IL-6 and IL-1ß secretion showing a net pro-inflammatory role for these receptors. Finally, we present the novel finding that THP-1 macrophages lacking the A2B receptor undergo pyroptosis when exposed to LPS, demonstrating a novel role of the A2B receptor in regulation of programmed cell death during inflammation. This work underscores the fundamental importance of adenosine signaling and provides insight into the independent roles of the adenosine receptors in modulating cytokine signaling.

Early evolutionary history (from bacteria to hemichordata) of the omnipresent purinergic signalling: A tribute to Geoff Burnstock inquisitive mind.

Purines and pyrimidines are indispensable molecules of life; they are fundamental for genetic code and bioenergetics. From the very early evolution of life purines have acquired the meaning of damage-associated extracellular signaller and purinergic receptors emerged in unicellular organisms. Ancestral purinoceptors are P2X-like ionotropic ligand-gated cationic channels showing 20-40% of homology with vertebrate P2X receptors; genes encoding ancestral P2X receptors have been detected in Protozoa, Algae, Fungi and Sponges; they are also present in some invertebrates, but are absent from the genome of insects, nematodes, and higher plants. Plants nevertheless evolved a sophisticated and widespread purinergic signalling system relying on the idiosyncratic purinoceptor P2K1/DORN1 linked to intracellular Ca2+ signalling. The advance of metabotropic purinoceptors starts later in evolution with adenosine receptors preceding the emergence of P2Y nucleotide and P0 adenine receptors. In vertebrates and mammals the purinergic signalling system reaches the summit and operates throughout all tissues and systems without anatomical or functional segregation.

G protein-coupled receptors expressed and studied in yeast. The adenosine receptor as a prime example.

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins with around 800 members in the human genome/proteome. Extracellular signals such as hormones and neurotransmitters regulate various biological processes via GPCRs, with GPCRs being the bodily target of 30-40% of current drugs on the market. Complete identification and understanding of GPCR functionality will provide opportunities for novel drug discovery. Yeast expresses three different endogenous GPCRs regulating pheromone and sugar sensing, with the pheromone pathway offering perspectives for the characterization of heterologous GPCR signaling. Moreover, yeast offers a ''null" background for studies on mammalian GPCRs, including GPCR activation and signaling, ligand identification, and characterization of disease-related mutations. This review focuses on modifications of the yeast pheromone signaling pathway for functional GPCR studies, and on opportunities and usage of the yeast system as a platform for human GPCR studies. Finally, this review discusses in some further detail studies of adenosine receptors heterologously expressed in yeast, and what Geoff Burnstock thought of this approach.

Increased Response to 3,4-Methylenedioxymethamphetamine (MDMA) Reward and Altered Gene Expression in Zebrafish During Short- and Long-Term Nicotine Withdrawal.

An interactive effect between nicotine and 3,4-methylenedioxymethamphetamine (MDMA) has been reported but the mechanism underlying such interaction is not completely understood. This study used zebrafish to explore gene expression changes associated with altered sensitivity to the rewarding effects of MDMA following 2-week exposure to nicotine and 2-60 days of nicotine withdrawal. Reward responses to MDMA were assessed using a conditioned place preference (CPP) paradigm and gene expression was evaluated using quantitative real-time PCR of mRNA from whole brain samples from drug-treated and control adult zebrafish. Zebrafish pre-exposed for 2 weeks to nicotine showed increased conditioned place preference in response to low-dose, 0.1 mg/kg, MDMA compared to un-exposed fish at 2, 7, 30 and 60 days withdrawal. Pre-exposure to nicotine for 2 weeks induced a significant increase of c-Fos and vasopressin receptor expression but a decrease of D3 dopaminergic and oxytocin receptor expression at 2 days of withdrawal. C-Fos mRNA increased also at 7, 30, 60 days of withdrawal. Nicotine pre-exposed zebrafish submitted to MDMA-induced CPP showed an increase in expression of p35 at day 2, α4 at day 30, vasopressin at day 7 and D3 dopaminergic receptor at day 7, 30 and 60. These gene alterations could account for the altered sensitivity to the rewarding effects of MDMA in nicotine pre-exposed fish, suggesting that zebrafish have an altered ability to modulate behaviour as a function of reward during nicotine withdrawal.

Pharmacological characterisation of novel adenosine A3 receptor antagonists.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

Bone Marrow and Adipose Tissue Adenosine Receptors Effect on Osteogenesis and Adipogenesis.

Adenosine is an extracellular signaling molecule that is particularly relevant in times of cellular stress, inflammation and metabolic disturbances when the levels of the purine increase. Adenosine acts on two G-protein-coupled stimulatory and on two G-protein-coupled inhibitory receptors, which have varying expression profiles in different tissues and conditions, and have different affinities for the endogenous ligand. Studies point to significant roles of adenosine and its receptors in metabolic disease and bone health, implicating the receptors as potential therapeutic targets. This review will highlight our current understanding of the dichotomous effects of adenosine and its receptors on adipogenesis versus osteogenesis within the bone marrow to maintain bone health, as well as its relationship to obesity. Therapeutic implications will also be reviewed.

Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels.

Progression of epithelial cancers predominantly proceeds by collective invasion of cell groups with coordinated cell-cell junctions and multicellular cytoskeletal activity. Collectively invading breast cancer cells express the gap junction protein connexin-43 (Cx43), yet whether Cx43 regulates collective invasion remains unclear. We here show that Cx43 mediates gap-junctional coupling between collectively invading breast cancer cells and, via hemichannels, adenosine nucleotide/nucleoside release into the extracellular space. Using molecular interference and rescue strategies, we identify that Cx43 hemichannel function, but not intercellular communication, induces leader cell activity and collective migration through the engagement of the adenosine receptor 1 (ADORA1) and AKT signaling. Accordingly, pharmacological inhibition of ADORA1 or AKT signaling caused leader cell collapse and halted collective invasion. ADORA1 inhibition further reduced local invasion of orthotopic mammary tumors in vivo, and joint up-regulation of Cx43 and ADORA1 in breast cancer patients correlated with decreased relapse-free survival. This identifies autocrine purinergic signaling, through Cx43 hemichannels, as a critical pathway in leader cell function and collective invasion.

Ticagrelor Prevents Endothelial Cell Apoptosis through the Adenosine Signalling Pathway in the Early Stages of Hypoxia.

BACKGROUND: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia-reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. METHOD: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. RESULTS: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. CONCLUSION: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium.

Adenosine Receptor Modulates Permissiveness of Baculovirus (Budded Virus) Infection via Regulation of Energy Metabolism in Bombyx mori.

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

Purinergic signaling, DAMPs, and inflammation.

Danger sensing is one of the most fundamental evolutionary features enabling multicellular organisms to perceive potential threats, escape from risky situations, fight actual intruders, and repair damage. Several endogenous molecules are used to "signal damage," currently referred to as "alarmins" or "damage-associated molecular patterns" (DAMPs), most being already present within all cells (preformed DAMPs), and thus ready to be released, and others neosynthesized following injury. Over recent years it has become overwhelmingly clear that adenosine 5'-triphosphate (ATP) is a ubiquitous and extremely efficient DAMP (thus promoting inflammation), and its main metabolite, adenosine, is a strong immunosuppressant (thus dampening inflammation). Extracellular ATP ligates and activates the P2 purinergic receptors (P2Rs) and is then degraded by soluble and plasma membrane ecto-nucleotidases to generate adenosine acting at P1 purinergic receptors (P1Rs). Extracellular ATP, P2Rs, ecto-nucleotidases, adenosine, and P1Rs are basic elements of the purinergic signaling network and fundamental pillars of inflammation.