Shufeng Jiedu capsule inhibits inflammation and apoptosis by activating A2AAR and inhibiting NF-κB to alleviate LPS-induced ALI.

ETHNOPHARMACOLOGICAL RELEVANCE: Shufeng Jiedu capsule (SFJDC) is a pure form of traditional Chinese medicine (TCM) that contains eight medicinal plants. Known for its anti-inflammatory and antipyretic effects, it is mostly used to treat upper respiratory tract infections and other infectious diseases, such as colds, pharyngitis, laryngitis, and tonsillitis. Both acute lung injury (ALI) and COVID-19 are closely related to lung damage, primarily manifesting as lung inflammation and epithelial cell damage. However, whether SFJDC can improve ALI and by what mechanism remain unclear. The purpose of this study was to explore whether SFJDC could be used as a prophylactic treatment for COVID-19 by improving acute lung injury. AIM OF THE STUDY: The purpose of this study was to determine whether SFJDC could protect against ALI caused by lipopolysaccharide (LPS), and we wanted to determine how SFJDC reduces inflammation and apoptosis pharmacologically and molecularly. MATERIALS AND METHODS: Preadministering SFJDC at 0.1 g/kg, 0.3 g/kg, or 0.5 g/kg for one week was followed by 5 mg/kg LPS to induce ALI in mice. Observations included the study of lung histomorphology, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) secretion, as well as the ratio of lung wet/dry weights. In addition, RAW264.7 cells were treated for 24 h with 1 µg/mL LPS after being pretreated for 1 h with 0.5 mg/mL SFJDC. In the samples, we detected TNF-α, IL-1ß, and IL-6. Cell apoptosis was detected by stimulating A549 cells for 24 h with RAW264.7 supernatant. Both in vitro and in vivo, the levels of A2A adenosine receptor (A2AAR), PKA, IκB, p-IκB, NF-κB P65 (P65), p-NF-κB P65 (p-P65), cleaved caspases-3 (Cc3), Bcl-2 associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) proteins were determined using Western blot analysis. RESULTS: Lung tissue morphology was improved as SFJDC decreased cytokine secretion, the ratio of lung wet/dry weights, and lung tissue secretion of proinflammatory cytokines. The expression of A2AAR was increased by SFJDC, and the phosphorylation of NF-κB was inhibited. TUNEL staining and flow cytometry showed that SFJDC inhibited apoptosis by reducing the expression of Cc3 and the ratio of Bax/Bcl-2. CONCLUSIONS: According to the results of this study, SFJDC can reduce inflammation and inhibit apoptosis. A2AAR activation and regulation of NF-κB expression are thought to make SFJDC anti-inflammatory and anti-apoptotic. A wide range of active ingredients may result in an anti-inflammatory and antipyretic effect with SFJDC.

Role of Caffeine in the Age-related Neurodegenerative Diseases: A Review.

Caffeine, a simple purine alkaloid with the proper chemical name 1,3,7-trimethylpurine- 2,6-dione, is an abundant compound present in coffee, food and drugs. It interacts with various pathways of which antagonism of adenosine receptors is the most significant but the other physiological pathways can be influenced by caffeine as well. Interaction with glutamate and dopamine neurotransmission pathways, competition with other substrates on cytochrome P450, non-competitive inhibition of acetylcholinesterase, blocking of nicotinic acetylcholine receptor and competitive inhibition of cyclic nucleotide phosphodiesterase can be mentioned. Because of caffeine availability in foods, beverages and drugs, it has practical relevance even if the effect is weak. Intake of coffee containing edibles for a long period or even for a substantial part of life makes caffeine´s impact significant. Low acute and chronic toxicity of caffeine is another important specification. The discoveries from the last few years point to the fact that caffeine would interfere with the progression of some age-related neurodegenerative disorders like Alzheimer's and Parkinson's diseases and dementia with Lewy bodies. In this review article, the recent findings about caffeine´s impact on neurodegenerative diseases are presented and important facts about the caffeine effect, including the substantial discoveries, are described.

Inosine mitigated diabetic peripheral neuropathy via modulating GLO1/AGEs/RAGE/NF-κB/Nrf2 and TGF-ß/PKC/TRPV1 signaling pathways.

Inosine is a dietary supplement that is widely used for managing numerous central neurological disorders. Interestingly, recent experimental investigation of inosine revealed its potential to promote peripheral neuroprotection after sciatic nerve injury. Such investigation has guided the focus of the current study to expose the potential of inosine in mitigating diabetic peripheral neuropathy (DPN) in rats and to study the possible underlying signaling pathways. Adult male Wistar rats were arbitrarily distributed into four groups. In the first group, animals received saline daily for 15 days whereas rats of the remaining groups received a single injection of both nicotinamide (50 mg/Kg/i.p.) and streptozotocin (52.5 mg/Kg/i.p.) for DPN induction. Afterward, inosine (10 mg/Kg/p.o.) was administered to two groups, either alone or in combination with caffeine (3.75 mg/Kg/p.o.), an adenosine receptor antagonist. As a result, inosine showed a hypoglycemic effect, restored the sciatic nerve histological structure, enhanced myelination, modulated conduction velocities and maintained behavioral responses. Furthermore, inosine increased GLO1, reduced AGE/RAGE axis and oxidative stress which in turn, downregulated NF-κB p65 and its phosphorylated form in the sciatic nerves. Inosine enhanced Nrf2 expression and its downstream molecule HO-1, resulting in increased CAT and SOD along with lowered MDA. Moreover, pain was relieved due to suppression of PKC and TRPV1 expression, which ultimately lead to reduced SP and TGF-ß. The potential effects of inosine were nearly blocked by caffeine administration; this emphasizes the role of adenosine receptors in inosine-mediated neuroprotective effects. In conclusion, inosine alleviated hyperglycemia-induced DPN via modulating GLO1/AGE/RAGE/NF-κB p65/Nrf2 and TGF-ß/PKC/TRPV1/SP pathways.

Gamma-decanolactone: Preliminary evaluation as potential antiparkinsonian drug.

Treatment of Parkinson's disease (PD) includes the use of monoamine oxidase-B (MAO-B) inhibitor drugs. In this work we have evaluated the possible gamma-decanolactone (GD) effect in vitro to inhibit the A and B isoforms of human monoamine oxidase (hMAO) enzyme and their citotoxicity in human hepatoma cell line (HepG2). Also, binding studies to A1, A2A A2B and A3 adenosine receptors were performed. A docking study of gamma-decanolactone has been carried out with the molecular targets of MAO-A and MAO-B isoforms. The physicochemical properties and ability to cross physiological barriers, as the blood brain barrier (BBB), was elucidated by computational studies. The in vivo assays, the rota-rod test, body temperature assessment and open field test were performed in reserpinized mice (1.5 mg/kg, i.p.; 18:00 before) to evaluate the effect of gamma-decanolactone (300 mg/kg), alone or associated with Levodopa plus Benserazide (LD + BZ, 100:25 mg/kg, i.p.). Gamma-decanolactone inhibited preferentially the MAO-B in a reversible manner, with an inhibitory concentration of 50% (IC50) 55.95 ± 9.06 µM. It was shown to be a safe drug since only at the highest concentration decreased the viability of HepG2 cells. It also does not bind to adenosine receptors investigated in this study. The molecular docking study show that the gamma-decanolactone ligand adopts a relatively compact conformation in the active site of hMAO-B, while we note an extended conformation of gamma-decanolactone ligand in the hMAO-A isoform. The physicochemical properties obtained, and the theoretical models utilized for the evaluation of ability to cross the BBB, predict a good gamma-decanolactone bioavailability and access to the central nervous system (CNS). In the in vivo studies, gamma-decanolactone partially reversed the ataxia of the reserpinized mice at 01:00 h and 01:30 h post-administration. Concomitant treatment of gamma-decanolactone with LD + BZ, at 01:30 h showed a potentiation of the reversibility of ataxia and facilitated the reversal of hypothermia caused by reserpine for all measured times (P <0.01 vs vehicle), except at 24:00 h, but not reversed the hypokinesia in the open field test. In summary, the results herein obtained and in conjunction with previous studies, suggest that gamma-decanolactone could be a drug with potential utility as antiparkinsonian drug.

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.

Electroacupuncture improves myocardial ischemia injury via activation of adenosine receptors.

Electroacupuncture (EA) can improve myocardial ischemia (MI) injury; nevertheless, the mechanism is not entirely clear. And there were disagreements about whether the effect of EA at acupoint in disease-affected meridian is better than EA at acupoint in non-affected meridian and sham acupoint. Here, we showed that the effect of EA at Neiguan (PC6) is better than EA at Hegu (LI4) and sham acupoint in affecting RPP and ECG, increasing ATP and ADO production, decreasing AMP production, and upregulating the mRNA expression levels of A1AR, A2aAR, and A2bAR; knockdown of A1AR or A2bAR reversed the effect of EA at PC6 in alleviating MI injury; knockdown of A2aAR had no influence on the cardiac protection of EA at PC6; thus, the cardioprotective effect of EA at PC6 needs A1AR and A2bAR, instead of A2aAR; considering that the cardio protection of adenosine receptor needs activation of other adenosine receptors, one of the reasons may be that after silence of A1AR or A2bAR, EA at PC6 could not impact the expression levels of the other two adenosine receptors, and after silence of A2aAR, EA at PC6 could impact the expression levels of A1AR and A2bAR. These results suggested that EA at PC6 may be a potential and effective treatment for MI by activation of A1AR and A2bAR.

Electroacupuncture inhibits visceral pain via adenosine receptors in mice with inflammatory bowel disease.

To investigate the involvement of peripheral adenosine receptors in the effect of electroacupuncture (EA) on visceral pain in mice with inflammatory bowel disease (IBD). 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was used to induce the visceral pain model. EA (1 mA, 2 Hz, 30 min) treatment was applied to bilateral acupoints "Dachangshu" (BL25) 1 day after TNBS injection once daily for 7 consecutive days. Von Frey filaments were used to measure the mechanical pain threshold. Western blot was used to detect the protein expression levels of adenosine 1 receptor (A1R), adenosine 2a receptor (A2aR), adenosine 2b receptor (A2bR), adenosine 3 receptor (A3R), substance P (SP), and interleukin 1 beta (IL-1ß) in colon tissue. EA significantly ameliorated the disease-related indices and reduced the expression of SP and IL-1ß in the colon tissues of mice with IBD. EA increased the expression of A1R, A2aR, and A3R and decreased the expression of A2bR in the colon tissue. Furthermore, the administration of adenosine receptor antagonists influenced the effect of EA. EA can inhibit the expression of the inflammatory factors SP and IL-1ß by regulating peripheral A1, A2a, A2b, and A3 receptors, thus inhibiting visceral pain in IBD mice.

Caffeine Treatment Promotes Differentiation and Maturation of Hypoxic Oligodendrocytes via Counterbalancing Adenosine 1 Adenosine Receptor-Induced Calcium Overload.

BACKGROUND We aimed to explore the involvement of adenosine 1 adenosine receptor (A1AR) in hypoxia-induced poor differentiation of oligodendrocytes (OLs), and the underlying mechanism of caffeine treatment in hypoxic injuries. MATERIAL AND METHODS Real-time polymerase chain reaction (RT-PCR) was used to assess the alterations of AR expression in cultured hypoxic OLs with or without caffeine treatment. Then, intracellular alterations of Ca²âº concentrations ([Ca²âº) were detected by confocal Fluo-3 imaging. The subsequent changes of myelin related protein expression were determined by western blot and immunofluorescence. RESULTS Three hours after hypoxia, significantly upregulated expression of A1AR was observed, accompanied with significantly decreased expression of oligodendrocyte transcription factor (Olig2). In addition, either hypoxia stimulation or 100 µM adenosine induced apparent elevation of resting [Ca²âº] in cultured OLs. However, pretreatment with DPCPX (A1AR selective antagonist) or caffeine abolished the [Ca²âº] increase, and the subsequent adenosine of high dose induced Ca²âº activity in developing OLs. Furthermore, caffeine or DPCPX improved the expression MBP and CNPase proteins after hypoxia stimulation, which resulted in the morphological maturation of OLs. CONCLUSIONS Caffeine treatment exerted protective effects on neonatal hypoxia injuries. It prevented Ca²âº overload injury, kept Ca²âº homeostasis in hypoxic developing OLs, and facilitated optimal expression of myelin related proteins by inhibiting A1AR in vitro. This study also provided experimental evidence for clinical application of caffeine in early treatment of neonatal hypoxia, and highlighted the potential significance of A1AR in anti-hypoxic drug discovery.

Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs.

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Coffee, caffeine, chlorogenic acid, and the purinergic system.

Coffee is a drink prepared from roasted coffee beans and is lauded for its aroma and flavour. It is the third most popular beverage in the world. This beverage is known by its stimulant effect associated with the presence of methylxanthines. Caffeine, a purine-like molecule (1,3,7 trymetylxantine), is the most important bioactive compound in coffee, among others such as chlorogenic acid (CGA), diterpenes, and trigonelline. CGA is a phenolic acid with biological properties as antioxidant, anti-inflammatory, neuroprotector, hypolipidemic, and hypoglicemic. Purinergic system plays a key role inneuromodulation and homeostasis. Extracellular ATP, other nucleotides and adenosine are signalling molecules that act through their specific receptors, namely purinoceptors, P1 for nucleosides and P2 for nucleotides. They regulate many pathological processes, since adenosine, for instance, can limit the damage caused by ATP in the excitotoxicity from the neuronal cells. The primary purpose of this review is to discuss the effects of coffee, caffeine, and CGA on the purinergic system. This review focuses on the relationship/interplay between coffee, caffeine, CGA, and adenosine, and their effects on ectonucleotidases activities as well as on the modulation of P1 and P2 receptors from central nervous system and also in peripheral tissue.

Ilex paraguariensis modulates fat metabolism in Caenorhabditis elegans through purinergic system (ADOR-1) and nuclear hormone receptor (NHR-49) pathways.

Ilex paraguariensis is a well-known plant that is widely consumed in South America, primarily as a drink called mate. Mate is described to have stimulant and medicinal properties. Considering the potential anti-lipid effects of I. paraguariensis infusion, we used an extract of this plant as a possible modulator of fat storage to control lipid metabolism in worms. Herein, the I. paraguariensis-dependent modulation of fat metabolism in Caenorhabditis elegans was investigated. C. elegans were treated with I. paraguariensis aqueous extract (1 mg/ml) from L1 larvae stage until adulthood, to simulate the primary form of consumption. Expression of adipocyte triglyceride lipase 1 (ATGL-1) and heat shock protein 16.2, lipid accumulation through C1-BODIPY-C12 (BODIPY) lipid staining, behavioral parameters, body length, total body energy expenditure and overall survival were analyzed. Total body energy expenditure was determined by the oxygen consumption rate in N2, nuclear hormone receptor knockout, nhr-49(nr2041), and adenosine receptor knockout, ador-1(ox489) strains. Ilex paraguariensis extract increased ATGL-1 expression 20.06% and decreased intestinal BODIPY fat staining 63.36%, compared with the respective control group, without affecting bacterial growth and energetic balance, while nhr-49(nr2041) and ador-1(ox489) strains blocked the worm fat loss. In addition, I. paraguariensis increased the oxygen consumption in N2 worms, but not in mutant strains, increased N2 worm survival following juglone exposure, and did not alter hsp-16.2 expression. We demonstrate for the first time that I. paraguariensis can decrease fat storage and increase body energy expenditure in worms. These effects depend on the purinergic system (ADOR-1) and NHR-49 pathways. Ilex paraguariensis upregulated the expression of ATGL-1 to modulate fat metabolism. Furthermore, our data corroborates with other studies that demonstrate that C. elegans is a useful tool for studies of fat metabolism and energy consumption.

Coffee consumption and reduced risk of developing type 2 diabetes: a systematic review with meta-analysis.

Context: Type 2 diabetes (T2D) is a major health problem worldwide that is associated with increased morbidity and mortality. There is increased interest in the value of different nutrition-based strategies for preventing the development of T2D. Objective: This review aims to cover current knowledge regarding the effects of coffee consumption on development of T2D or modulation of adverse complications. A meta-analysis on coffee consumption and the risk of T2D was conducted. Moreover, bioactive components in coffee, polymorphisms, and potential underlying mechanism(s) in relation to T2D and adverse complications are discussed. Data sources: PubMed was searched up to December 1, 2017, and prospective cohort and nested case-control studies of the association between coffee consumption and T2D risk were selected. Data extraction: Two investigators independently extracted data from included studies. Results: A total of 30 prospective studies with 1 185 210 participants and 53 018 incident T2D cases were included in the meta-analysis. The pooled relative risk (RR) was 0.71 (95% confidence interval [CI], 0.67-0.76) for the highest category of coffee consumption (median consumption, 5 cups/d) vs the lowest category (median consumption, 0 cups/d). The risk of T2D decreased by 6% (RR = 0.94; 95%CI, 0.93-0.95) for each cup-per-day increase in coffee consumption. Results were similar for caffeinated coffee consumption (per additional cup of coffee per day: RR = 0.93; 95%CI, 0.90-0.96) and decaffeinated coffee consumption (corresponding RR = 0.94; 95%CI, 0.90-0.98). Conclusions: Available evidence indicates that coffee consumption is inversely associated with risk of T2D. Possible mechanisms behind this association include thermogenic, antioxidative, and anti-inflammatory effects; modulation of adenosine receptor signaling; and microbiome content and diversity.

[Effects of electroacupuncture on the expression of adenosine receptors in the heart tissue of myocardial ischemia rats].

OBJECTIVE: To explore the impact of electroacupuncture (EA) on the protein expression of adenosine receptors in the heart of the rats with myocardial ischemia (MI). METHODS: Thirty healthy male SD rats were divided randomly into a control group (n=6), a model group (n=12) and an EA group (n=12). We ligated the left anterior descending artery (LAD) for MI model in the model group and EA group, and exposed the heart after opening the chest without ligation in the control group. EA, 2 Hz /15 Hz and 1.5-2 mA, was applied at bilateral"Neiguan"(PC 6) in the EA group for 20 min, once a day for continuous 5 days. No intervention except grabbing and fixation was used in the control group and model group. We applied 2% TTC staining to observe the infarct size of myocardium, colorimetry to analyze serum lactic dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), radio-immunity assessment to detect cardiac troponin T (cTnT), Western blot to evaluate the adenosine A1 receptor (A1AR), A2aAR, A2bAR and A3AR. RESULTS: After treatment, myocardial infarction of (27.56±3.24)% was obvious in the model group; the myocardial infarction in the EA group was (21.04±3.61)%, with statistical significance (P<0.05). The expressions of serum LDH, CK, CK-MB and cTnT levels in the model group increased compared with those in the control group (all P<0.01), and the expressions of LDH, CK, CK-MB and cTnT levels in the EA group decreased compared with those in the model group (P<0.05，P<0.01). The A1AR expression in the model group was not different from that in the control group (P>0.05), and A2aAR、A2bAR、A3AR expressions decreased (P<0.05, P<0.01). A2aAR and A2bAR expressions in the EA group increased compared with those in the model group (both P<0.01), and there was no statistical significance between A1AR and A3AR expressions (both P>0.05). . CONCLUSION: EA may achieve cardioprotective effect by regulating the expressions of A2aAR and A2bAR in myocardial tissue, which induce the corresponding signal cascade for reducing myocardial infarction area.

Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode.

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A2A and A1ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A2A/A3, ARs, or A3AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A2A/A3, A1/A3, A1/A2A/A3, and A3). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A3 receptor.

[Effect of Electroacupuncture on Adenosine Receptor Expression in White Adipose Tissue of Diet-induced Obese Mice].

OBJECTIVE: To investigate the effects of electroacupuncture (EA) on the expression of adenosine receptor (AR) in the white adipose tissue (WAT) of diet-induced obese (DIO) mice, so as to reveal a peripheral mechanism of EA underlying improvement of body weight. METHODS: Forty three-week-old C 57 BL/6 male mice were divided into normal diet group (n=12) and high fat diet group (n=28) randomly, and fed by normal diet and high fat diet for 12 weeks, respectively. In the high fat diet group, mice with body weight over 20% heavier than that of the normal diet group were considered as obese mice. The normal diet mice and the obese mice were divided into normal group (CD, n=5) and normal plus EA group (CD+EA, n=7), or obese group (HFD, n=6) and obese plus EA group (HFD+EA, n=12). The CD+EA group and the HFD+EA group were treated with EA at "Zusanli"(ST 36) and "Neiting"(ST 44, 2 Hz/15 Hz, 0.6-1.0 mA) for 20 min, 6 times a week for 4 weeks. Body weight, ratio of WAT/body weight were calculated, qPCR and Western blot were applied to detect mRNA and protein levels of adenosine receptors in the epididymal adipose tissue (Epi-WAT), respectively. RESULTS: Compared with the normal diet group, high fat diet significantly increased body weight in C 57 BL/6 mice after feeding for 12 weeks (P<0.01); 18 out of 28 mice in the high fat diet group were classified as obesity. After treatment, the body weight and the ratio of Epi-WAT/body weight of the HFD group were increased than those in the CD group (P<0.05), the change of body weight in the HFD group was bigger than that in the CD group (P<0.01). Compared with the HFD group, the body weight and the ratio of Epi-WAT/body weight of the HFD+EA group were decreased after EA (P<0.05), the change of body weight was also significantly increased (P<0.01). No significant differences were found among the four groups in the expression level of A1R mRNA (P>0.05). The expression of A3R mRNA in the HFD group was lower than that in the CD group (P<0.01), while the expressions of A2A R and A2BR proteins were decreased in the HFD group than in the CD group (P<0.01). In comparison with the HFD group, the expression levels of A2AR and A2BR mRNAs and proteins were significantly up-regulated in the HFD+EA group, respectively (P<0.05, P<0.01). CONCLUSIONS: EA intervention is able to reduce the body weight of DIO mice, which Feb be associated with its effects in regulating the expression of A2AR and A2BR in WAT, suggesting a new mechanism of EA in accelerating peripheral WAT metabolism.

Caffeine May Reduce Perceived Sweet Taste in Humans, Supporting Evidence That Adenosine Receptors Modulate Taste.

Multiple recent reports have detailed the presence of adenosine receptors in sweet sensitive taste cells of mice. These receptors are activated by endogenous adenosine in the plasma to enhance sweet signals within the taste bud, before reporting to the primary afferent. As we commonly consume caffeine, a powerful antagonist for such receptors, in our daily lives, an intriguing question we sought to answer was whether the caffeine we habitually consume in coffee can inhibit the perception of sweet taste in humans. 107 panelists were randomly assigned to 2 groups, sampling decaffeinated coffee supplemented with either 200 mg of caffeine, about the level found in a strong cup of coffee, or an equally bitter concentration of quinine. Participants subsequently performed sensory testing, with the session repeated in the alternative condition in a second session on a separate day. Panelists rated both the sweetened coffee itself and subsequent sucrose solutions as less sweet in the caffeine condition, despite the treatment having no effect on bitter, sour, salty, or umami perception. Panelists were also unable to discern whether they had consumed the caffeinated or noncaffeinated coffee, with ratings of alertness increased equally, but no significant improvement in reaction times, highlighting coffee's powerful placebo effect. This work validates earlier observations in rodents in a human population.

Caffeine inhibits hypothalamic A1R to excite oxytocin neuron and ameliorate dietary obesity in mice.

Caffeine, an antagonist of the adenosine receptor A1R, is used as a dietary supplement to reduce body weight, although the underlying mechanism is unclear. Here, we report that adenosine level in the cerebrospinal fluid, and hypothalamic expression of A1R, are increased in the diet-induced obesity (DIO) mouse. We find that mice with overexpression of A1R in the neurons of paraventricular nucleus (PVN) of the hypothalamus are hyperphagic, have glucose intolerance and high body weight. Central or peripheral administration of caffeine reduces the body weight of DIO mice by the suppression of appetite and increasing of energy expenditure. We also show that caffeine excites oxytocin expressing neurons, and blockade of the action of oxytocin significantly attenuates the effect of caffeine on energy balance. These data suggest that caffeine inhibits A1Rs expressed on PVN oxytocin neurons to negatively regulate energy balance in DIO mice.

A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development.

Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.

Extracellular ATP and adenosine: The Yin and Yang in immune responses?

Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses.