Journal: International journal of molecular sciences

Article Title: Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A 1 and A 2A Receptors.

doi: 10.3390/ijms21239281

Figure Lengend Snippet: Figure 2. Functional competition between GUO and adenosine (ADO) or selective agonists for A1R and A2AR on GUO-mediated anxiolytic-like effect evaluated by EPM test during a 5 min session. Pretreatment with ADO (10 mg/kg or 30 mg/kg) resulted in a complete block of anxiolytic-like effect of GUO (30 mg/kg) with significant decrease in (A) number of open arm entries [F(5,62) =6.972, p < 0.0001], (B) time spent in open arms [F(5,62) = 6.566, p < 0.0001] and (C) the ratio of time in open arm and total time in arms [F(5,62) = 18.33, p < 0.0001], when compared to GUO treated group. Pretreatment with A1R agonist CPA (0.1 mg/kg) or with A2AR agonist CGS (0.4 mg/kg) was able to completely block the anxiolytic-like effect of GUO (30 mg/kg) with significant decrease in (D) number of open arm entries [F(5,60) = 5.450, p < 0.0005], (E) time spent in open arms [F(5,60) = 22.29, p < 0.0001] and (F) the ratio of time in open arm and total time in arms [F(5,60) = 15.54, p < 0.0001], when compared to GUO treated group. CGS alone produced a significant reduction in time spent in open arms and ratio of time in open arm and total time in arms as compared to control group (Ctrl): each bar represents the mean value ± SD. Tukey test: * p < 0.05, ** p < 0.01; *** p < 0.001. Ctrl raw mean values: (A) 6.23; (B) 76.23 s; (C) 0.33; (D) 7; (E) 97.88 s; (F) 0.43.

Article Snippet: The following drugs were used: GUO (G6264 Sigma-Aldrich St. Louis, MO, USA) and nonselective ARs ligand ADO (A3377 Sigma-Aldrich St. Louis, MO, USA) were dissolved in 0.9% physiological saline solution (pH 9.0); selective A1R agonists N6-Cyclopentyladenosine (CPA, C8031 Sigma-Aldrich) and 2-Chloro-N6-cyclopentyladenosine (CCPA, 1705 Tocris) or A2AR agonist 2-p-(2- Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680 hydrocloride, sc-211062 Santa Cruz Biotechnology) as well as selective A1R antagonist 8-Cyclopentyl-1,3dipropylxanthine (DPCPX, sc-200115 Santa-Cruz Biotechnology) or A2AR antagonist 4-(-2-[7-amino2-{2-furyl}triazolo [57] triazin-5-yl-amino]ethyl)phenol (ZM241385, Z0153, Sigma-Aldrich) were first dissolved in DMSO and thereafter in 0.9% physiological saline solution; the ARs nonselective antagonist caffeine (C0750, Sigma-Aldrich) was dissolved in 0.9% physiological saline solution.

Techniques: Functional Assay, Blocking Assay, Produced, Control

Journal: International journal of molecular sciences

Article Title: Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A 1 and A 2A Receptors.

doi: 10.3390/ijms21239281

Figure Lengend Snippet: Figure 3. Effects of A1R and A2AR antagonists on anxiolytic-like effect of GUO evaluated by EPM test during a 5 min session. (A–C) Functional competition between GUO and nonselective ARs antagonist caffeine (Caff). Caffeine alone (30 mg/kg) induced anxiolytic effects, as shown by significant increase in time spent in open arms and ratio of time in open arms and total time in arms as compared to control group (Ctrl). Pretreatment with caffeine (30 mg/kg) did not block anxiolytic-like effect of GUO and showed significant increase in time in open arms and ratio of time in open arms and total time in arms as compared to control group. (A–C) Functional competition between caffeine (30 mg/kg) and ADO (30 mg/kg) showed preservation of caffeine anxiolytic effect with significant increase in time spent in open arms and ratio of time in open arms and total time in arms as compared to control group. (D–F) Functional competition between GUO and selective AR antagonists. Selective A1R antagonist DPCPX (1 mg/kg) was not able to block GUO anxiolytic-like effect, as shown by significant increase in time in open arms and ratio of time in open arms and total time in arms as compared to control group. DPCPX alone did not show significant changes of all EPM parameters as compared to control group. Pretreatment with selective A2AR antagonist ZM241385 (0.4 mg/kg) was not able to block GUO (30 mg/kg) anxiolytic-like effect, as shown by significant increase in time in open arms and ratio of time in open arms and total time in arms as compared to control group, and ZM241385 alone did not show

Article Snippet: The following drugs were used: GUO (G6264 Sigma-Aldrich St. Louis, MO, USA) and nonselective ARs ligand ADO (A3377 Sigma-Aldrich St. Louis, MO, USA) were dissolved in 0.9% physiological saline solution (pH 9.0); selective A1R agonists N6-Cyclopentyladenosine (CPA, C8031 Sigma-Aldrich) and 2-Chloro-N6-cyclopentyladenosine (CCPA, 1705 Tocris) or A2AR agonist 2-p-(2- Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680 hydrocloride, sc-211062 Santa Cruz Biotechnology) as well as selective A1R antagonist 8-Cyclopentyl-1,3dipropylxanthine (DPCPX, sc-200115 Santa-Cruz Biotechnology) or A2AR antagonist 4-(-2-[7-amino2-{2-furyl}triazolo [57] triazin-5-yl-amino]ethyl)phenol (ZM241385, Z0153, Sigma-Aldrich) were first dissolved in DMSO and thereafter in 0.9% physiological saline solution; the ARs nonselective antagonist caffeine (C0750, Sigma-Aldrich) was dissolved in 0.9% physiological saline solution.

Techniques: Functional Assay, Control, Blocking Assay, Preserving

Journal: International journal of molecular sciences

Article Title: Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A 1 and A 2A Receptors.

doi: 10.3390/ijms21239281

Figure Lengend Snippet: Figure 5. [3H]GUO binding to hippocampal membranes. (A) The saturation isotherm studies showed that the binding became saturable at [3H]GUO concentrations ranging between 100 and 300 nM. The pooled data resolved for the presence of a single high affinity binding site with an apparent KD = 80 ± 34 nM; Bmax= 2339 ± 339 fmol/mg /protein. (B) Displacement of [3H]GUO binding by GUO and nonselective ARs agonist ADO in rat hippocampal membranes. Competition binding between GUO and ADO showed for ADO almost the same potency order of GUO to displace [3H]GUO (pIC50 6.069 ± 0.2074 and pIC50 −6.251 ± 0.1649, respectively), although ADO was able to displace only 70% of [3H]GUO binding. (C) [3H]GUO displacement (70 nM) by 500 µM of GUO, ADO, caffeine and selective agonists CPA or CGS21680 in rat hippocampal membranes.ADO was almost as effective as GUO in displacing [3H]GUO binding. Selective A1R agonist CPA or selective A2AR agonist CGS21680 displaced respectively 57% and 11% of [3H]GUO binding. Nonselective ARs antagonist caffeine displaced only 24% of [3H]GUO binding.

Article Snippet: The following drugs were used: GUO (G6264 Sigma-Aldrich St. Louis, MO, USA) and nonselective ARs ligand ADO (A3377 Sigma-Aldrich St. Louis, MO, USA) were dissolved in 0.9% physiological saline solution (pH 9.0); selective A1R agonists N6-Cyclopentyladenosine (CPA, C8031 Sigma-Aldrich) and 2-Chloro-N6-cyclopentyladenosine (CCPA, 1705 Tocris) or A2AR agonist 2-p-(2- Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680 hydrocloride, sc-211062 Santa Cruz Biotechnology) as well as selective A1R antagonist 8-Cyclopentyl-1,3dipropylxanthine (DPCPX, sc-200115 Santa-Cruz Biotechnology) or A2AR antagonist 4-(-2-[7-amino2-{2-furyl}triazolo [57] triazin-5-yl-amino]ethyl)phenol (ZM241385, Z0153, Sigma-Aldrich) were first dissolved in DMSO and thereafter in 0.9% physiological saline solution; the ARs nonselective antagonist caffeine (C0750, Sigma-Aldrich) was dissolved in 0.9% physiological saline solution.

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: The different effects of four adenosine receptors in liver fibrosis

doi: 10.3389/fphar.2024.1424624

Figure Lengend Snippet: NECA inhibited HSC activation and alleviated liver fibrosis. (A) Flowchart of animal experiments. (B,C) Serum ALT and AST levels in control, CCl 4 -induced mice, and NECA-treated mice. (D–F) Representative liver tissue sections of control, CCl 4 -, and NECA-treated mice were detected by HE, Masson, and Sirius Red staining. Scale bar: 50 μm; magnification: ×20. (G–J) qRT-PCR and IHC analysis showed the expression levels of α-SMA and Col1α1 in control, CCl 4 -, and NECA-treated mice. Scale bar: 50 μm; magnification: ×20. The positive area statistics of Masson and Sirius Red staining were measured using ImageJ software. (K,L) qRT-PCR analysis showed the expression levels of α-SMA and Col1α1 in LX2 cells. (M) Effect of NECA treatment on LX2 cell proliferation. (N) Expression of A1R, A2AR, A2BR, and A3R in LX2 cells with or without NECA treatment. * p < 0.05, ** p < 0.01, *** p < 0.0005, and **** p < 0.0001; ns: no significance.

Article Snippet: The mice were randomly divided into different groups: control group—olive oil (MACKLIN, China, Cat: 8001-25-0), 1 mL/kg i.p.; CCl 4- induced group [25% CCl 4 (MACKLIN, China, Cat: 56-23-5) (CCl 4 : olive oil = 1:3), 1 mL/kg i.p.]; NECA treatment group [1 mL/kg i.p. 25% CCl 4 plus 0.1 mg/kg i.p. NECA (GlpBio, Montclair, CA, United States, Cat: GC15304)]; A1R agonist group [1 mL/kg i.p. 25% CCl 4 plus 0.5 mg/kg i.p. CCPA (GlpBio, Montclair, CA, United States, Cat: GC45773)]; A2AR agonist group [1 mL/kg i.p. 25% CCl 4 plus 1 mg/kg i.p. CGS21680 (GlpBio, Montclair, CA, United States, Cat: GC10172)]; A2BR agonist group [1 mL/kg i.p. 25% CCl 4 plus 4 mg/kg i.p. BAY-606583 (TargetMol, China, Cat: 910487-58-0)]; and A3R agonist group [1 mL/kg i.p. 25% CCl 4 plus 200 μg/kg i.p. namodenoson (TargetMol, China, Cat: 163042-96-4)].

Techniques: Activation Assay, Control, Staining, Quantitative RT-PCR, Expressing, Software

Journal: Advanced Science

Article Title: Targeting Adenosine A2b Receptor Promotes Penile Rehabilitation of Refractory Erectile Dysfunction

doi: 10.1002/advs.202306514

Figure Lengend Snippet: Under normoxic condition, adenosine A2a receptor activation up‐regulates HIF‐1α, and down‐regulates PDE5. A) A7r5 cell viability under NECA stimulation. The cell viability was detected by the CCK‐8 test. B) Representative WB protein bands and statistical analysis of TGF‐β, Smad2/3, p‐Smad2/3, α‐SMA, and Tubulin. C) Relative mRNA expressions of HIF‐1α, PDE5, eNOS, and TGF‐β. D) Representative WB protein bands and statistical analysis of A1, A2a, A2b, and A3 receptors under NECA stimulation. E–H) Relative mRNA expressions of HIF‐1α, PDE5, eNOS, and TGF‐β were analyzed. The A7r5 cells were treated by NECA (3 and 10 µ m ) and adenosine receptor inhibitors (PSB‐36 for the A1 receptor, Istra for the A2a receptor, PSB‐603 for the A2b receptor, and MRS‐1523 for the A3 receptor). I) Representative WB protein bands and statistical analysis of HIF‐1α, PDE5, eNOS, TGF‐β, and Tubulin. The A7r5 cells were treated by NECA (10 µ m ) and A2a receptor inhibitors (Istra). Statistical analysis was performed using an unpaired t‐test or ANOVA. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: NECA and all the other inhibitors were purchased from ApexBIO (Houston, TX, USA).

Techniques: Activation Assay, CCK-8 Assay

Journal: Advanced Science

Article Title: Targeting Adenosine A2b Receptor Promotes Penile Rehabilitation of Refractory Erectile Dysfunction

doi: 10.1002/advs.202306514

Figure Lengend Snippet: Under hypoxia, adenosine A2b receptor activation up‐regulates HIF‐1α, and down‐regulates PDE5. A) A7r5 cell viability under NECA stimulation. B) Representative WB protein bands and statistical analysis of A1, A2a, A2b, and A3 receptors. C) Volcano plot visualizing the altered gene expression profiles under the stimulation of NECA. D) Cluster and heatmap of the differentially expressed genes. E–G) HIF‐1, cGMP‐PKG, and cAMP signaling pathways were significantly enriched by GSEA. H) Relative mRNA expressions of HIF‐1α, PDE5, and eNOS. I) Relative mRNA expressions of HIF‐1α, PDE5, and eNOS. The A7r5 cells were treated with NECA (10 µ m ) and adenosine receptor inhibitors. J) Representative WB protein bands and statistical analysis of HIF‐1α, PDE5, and eNOS. The A7r5 cells were treated by NECA (10 µ m ) and A2b receptor inhibitor (PSB‐603). K) Representative WB protein bands and statistical analysis of A2b receptor. The A7r5 cells were treated with adenosine receptor inhibitors. L) Representative WB protein bands and statistical analysis of α‐SMA. M): Representative WB protein bands and statistical analysis of α‐SMA. N) Representative WB protein bands and statistical analysis of HIF‐1α, α‐SMA, PDE5, and A2b receptor. The primary corpus cavernosum smooth muscle cells were treated by NECA (10 µ m ) and A2b receptor inhibitor (PSB‐603). O) Relative mRNA expressions of HIF‐1α, eNOS, PDE5, and A2b receptor. Statistical analysis was performed using an unpaired t‐test or ANOVA. N = 3 or 5. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: NECA and all the other inhibitors were purchased from ApexBIO (Houston, TX, USA).

Techniques: Activation Assay, Expressing

Journal: Cell Death & Disease

Article Title: The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

doi: 10.1038/cddis.2014.576

Figure Lengend Snippet: Adenosine induces IL-1 β secretion and ATP release in THP1 human macrophages. PMA-primed THP1 cells were stimulated during 6 h with 250 μ g/ml nano-SiO 2 , 500 μ g/ml nano-TiO 2 and/or high concentrations of adenosine (Ado), and eATP and IL-1 β releases were measured. Adenosine potentiated eATP release and IL-1 β secretion upon nanoparticle stimulation without inducing change in cell viability ( a ). High doses of adenosine alone induced IL-1 β secretion by PMA-primed THP1 in a dose-dependent manner ( b ). The caspase-1-specific inhibitor Z-YVAD-fmk (5 μ M) remarkably reduced adenosine- and/or nanoparticle-induced IL-1 β secretion; Z-YVAD-fmk alone had no effect ( c ). Adenosine-dependent induction of IL-1 β secretion was reduced in THP1 sh NLRP3 or THP1 sh ASC but not in unmodified THP1 or THP1 sh CTL ( d ). PMA-primed THP1 cells were stimulated with increasing doses of adenosine, the non-metabolisable analogue of adenosine NECA or inosine (Ino), the product of adenosine hydrolysis by ADA, and cell supernatants were analyzed to measure eATP, IL-1β and cell death ( e ). PMA-primed THP1 cells were stimulated with mM doses of adenosine in the presence of 5-iodotubercidin (5-Iodo), an inhibitor of both adenosine kinase and ENTs, or in the presence of NBMPR, an inhibitor of ENT1 at nM doses and ENT2 at μ M concentrations, and eATP and IL-1 β releases were measured at 6 h ( f , g ). Quantitative PCR analysis of ENT1, ENT2 and NLRP3 expression in PMA-primed THP1 stimulated for 6 h with increasing concentrations of Ado was performed ( h , i ). Intracellular ATP contents were measured 6 h after stimulation with high concentrations of adenosine ( j ). Data are representative of 2–3 independent experiments (* P ≤0.05, ** P ≤0.01, *** P ≤0.001, ns: not statistically different)

Article Snippet: Adenosine deaminase (ADA) (A5168), ADP, ADP β S, adenosine (Ado), apyrase grade VII (A6535), ARL67156, ATP γ S, ATP, carbenoxolone (Cbx), DPCPX, flufanemic acid (FFA), inosine (Ino), MRS1523, MRS1754, MRS2395, MRS2578, periodate-oxidised ATP (oATP), phorbol 12-myristate 13-acetate (PMA), SCH58261 and U73122 were from Sigma (St. Quentin Fallavier, France); suramin was from VWR (Fontenay-sous-bois, France); LPS (lipopolysaccharide from Escherichia coli , serotype 055:B5) was from Invivogen (Toulouse, France); 2-APB, 5-Iodotubercidin, Forskolin, MRS2500, NBMPR and Z-YVAD-fmk were from Tocris (Bristol, UK) and NECA and SQ22536 were from Merck Millipore (Nottingham, UK).

Techniques: Real-time Polymerase Chain Reaction, Expressing