Journal: Metabolites

Article Title: Urinary ATP Levels Are Controlled by Nucleotidases Released from the Urothelium in a Regulated Manner.

doi: 10.3390/metabo13010030

Figure Lengend Snippet: Figure 7. Effect of forskolin (FSK) on the hydrolysis of eATP by soluble enzymes released in bladder lumen during filling. Decrease of eATP (a) and increase in eADP (b), eAMP (c), and eADO (d) after addition of eATP in ILS from bladder preparations filled with vehicle (DMSO 0.1%, n = 7) or FSK (n = 4 each); n, number of bladder preparations. Note that the decrease of eATP and the formation of eADP and eADO were enhanced in the presence of FSK. Asterisks denote significant differences from vehicle controls. * p < 0.05, ** p < 0.01. 2 way ANOVA with Tukey’s multiple comparisons tests.

Article Snippet: Adenosine, ATP, ADP, AMP, dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), 6-[(3-aminophenyl)methyl]-N,N,5-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7amine (ENPP1 Inhibitor C) (Cayman Chemicals, Ann Arbor, MI, USA), (−)-pbromotetramisole oxalate (L-p-BT) (MedChemExpress, Monmouth Junction, NJ, USA); 6- N,N-Diethyl-D-β,γ-dibromomethyleneATP trisodium salt (ARL67156), sodium metatungstate (POM-1), and 1-Amino-4-(1-naphthyl)aminoanthraquinone-2-sulfonic acid sodium salt (PSB06126), FSK, 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) (BioTechne Tocris, Minneapolis, MN, USA).

Techniques:

Journal: BioMed Research International

Article Title: Protective Effects of Adiponectin against Cobalt Chloride-Induced Apoptosis of Smooth Muscle Cells via cAMP/PKA Pathway

doi: 10.1155/2020/7169348

Figure Lengend Snippet: cAMP and PKA inhibition blocked APN-mediated protection against CoCl 2 effects. (a, c–e) The CoCl 2 -induced reduction SMCs viability and SOD activity and increase apoptosis and MDA levels were blocked by APN; those effects of APN could be blocked by SQ22536 and H 89. (b) Statistical analysis of the rate of apoptosis in each group. (f) The CoCl 2 -induced Bax and cleaved caspase-3 upregulation and Bcl-2 downregulation were blocked by APN, and effects of APN could also be blocked by SQ22536 and H 89. (g) Statistical analysis of the expression of Bcl-2, Bax, and cleaved caspase-3. (h–j) Expression and location of Bcl-2, Bax, and caspase-3 (green) in the SMCs. Blue indicates nuclei as stained by DAPI. Scale bar: 25 μ m (∗ P < 0.05 vs. control, ∗∗ P < 0.01 vs. control, # P < 0.05 vs. CoCl 2 , ## P < 0.01 vs. CoCl 2 , & P < 0.05 vs. APN+CoCl 2 , n = 5, data shown as the mean ± SE). CoCl 2 : cobalt chloride; APN: adiponectin; SQ22536: cAMP inhibitor; H 89: PKA inhibitor; SOD: superoxide dismutase; MDA: malondialdehyde; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein.

Article Snippet: To elucidate the role of the cAMP/PKA signaling pathway, the cells were pretreated with cAMP inhibitor SQ22536 (#S8283, Selleck Chemicals, USA, 1 mM, 30 min) or PKA inhibitor H 89 (#S1582, Selleck Chemicals, USA, 50 μ M, 1 h) before being treated with CoCl 2 and APN.

Techniques: Inhibition, Activity Assay, Expressing, Staining, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: Prostaglandin E 2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

doi: 10.1152/ajpcell.00508.2009

Figure Lengend Snippet: Involvement of PI3K and cAMP/PKA signaling pathways in IL-6 mRNA synthesis and cAMP production induced by PGE2 or forskolin in human T/C28a2 chondrocytes. T/C-28a2 cells were incubated with either PGE2 (10 μM) (A, C) or forskolin (20 μM) (B, D) for 1 or 2 h in presence of the PI3K inhibitors [LY-294002 (30 μM) or wortmannin (10 μM)] or the PKA inhibitor H89 (10 μM) or the adenylyl cyclase inhibitor SQ-22536 (100 μM). IL-6 mRNA synthesis was determined by qRT-PCR (A, B). GAPDH served as internal control. cAMP formation was analyzed by a cAMP enzyme immunoassay kit (C, D). Data are means ± SE of three independent experiments. *P < 0.05 with respect to all pharmacological treatments and no treatment (vehicle) control. E: Western blots showing the effects of the phosphatidylinositol 3 kinase (PI3K) inhibitors (LY-294002; wortmannin), PKA inhibitor H89, or the adenylyl cyclase inhibitor SQ-22536 on PGE2- and forskolin-mediated phosphorylation of Akt (Ser473) and CREB (Ser133) using specific antibodies (Abs). Equal loading in each lane is shown by the similar intensities of Akt, CREB, and β-actin. The immunoblots shown are representative of three independent experiments with similar results. The intensity of bands was quantified relative to β-actin for each treatment using the Bio-Rad gel image system and then normalized with respect to the value obtained for the untreated control. Data are means ± SE of three independent experiments. *P < 0.05 with respect to no treatment control.

Article Snippet: PGE 2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

Techniques: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Prostaglandin E 2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

doi: 10.1152/ajpcell.00508.2009

Figure Lengend Snippet: PGE2 and forskolin induce NF-κB activation in human T/C28a2 chondrocytes via cAMP/PKA and PI3K signaling pathways. T/C-28a2 cells were treated with PGE2 (10 μM) (A) or forskolin (20 μM) (B) for the indicated time intervals. Nuclear extracts were then isolated, and NF-κB-specific DNA-protein complex formation was determined by EMSA. T/C-28a2 chondrocytes were treated for 2 h with either PGE2 (C, E, G, H) or forskolin (D, F, G, H) in the presence or absence of the PI3K inhibitors [LY-294002 (30 μM) or wortmannin (10 μM)], the PKA inhibitor H89 (10 μM), or the adenylyl cyclase inhibitor SQ-22536 (100 μM). Nuclear extracts were prepared for the determination of NF-κB-specific DNA-protein complex formation by EMSA (C, D). Supershift (E, F) assays using an anti-p65 Ab were carried out as outlined in materials and methods. Results of a competition experiment using 50-fold unlabeled NF-κB oligonucleotide (cold probe) are shown. Cross-linked chromatin was immunoprecipitated using an anti-p65 antibody (G, H). In ChIP assays, the anti-RNA polymerase II antibody was used as positive control, whereas the normal mouse IgG and anti-TLR4 antibodies were used as negative controls. DNA purified from both the immunoprecipitated (IP) and preimmune (input) specimens was subjected to PCR amplification using primers for the GAPDH (control) and p65 promoter genes. All experiments are representative of three independent experiments, all revealing similar results.

Article Snippet: PGE 2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

Techniques: Activation Assay, Isolation, Immunoprecipitation, Positive Control, Purification, Amplification

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

doi: 10.1155/2022/7941039

Figure Lengend Snippet: (a) Effect of SQ22536 and LY294002 on platelet aggregation. Representative images of CD62p expression (b) and PAC-1 activation (c) after different interventions. Quantitative analysis of CD62p expression (d) and PAC-1 activation (e) (MFI). Results are expressed as mean ± SD. n = 6. & P < 0.05 vs. resting; ∗ P < 0.05 vs. control; # P < 0.05 vs. model; + P < 0.05 vs. TMP; ▲ P < 0.05 vs. SQ22536; △ P < 0.05 vs. LY294002.

Article Snippet: SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA).

Techniques: Expressing, Activation Assay, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

doi: 10.1155/2022/7941039

Figure Lengend Snippet: Effect of SQ22536 and LY294002 on the release of sCD40L and IL-1 β . Results are expressed as mean ± SD. n = 6. & P < 0.05 vs. resting; ∗ P < 0.05 vs. control; # P < 0.05 vs. model; + P < 0.05 vs. TMP; ▲ P < 0.05 vs. SQ22536; △ P < 0.05 vs. LY294002.

Article Snippet: SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA).

Techniques: Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

doi: 10.1155/2022/7941039

Figure Lengend Snippet: (a, c) Effect of SQ22536 on cAMP release and p-VASP ser157 expression. Results are expressed as mean ± SD. n = 6. and P < 0.05 vs. resting; ∗ P < 0.05 vs. control; # P < 0.05 vs. model; + P < 0.05 vs. TMP. (b) Effect of SQ22536 on p-VASP ser157 expression various groups evaluated using western blotting.

Article Snippet: SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA).

Techniques: Expressing, Control, Western Blot

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: Nanoparticles trigger NLRP3 inflammasome through activation of PLC-InsP3 and inhibition of ADCY-cAMP pathways. LPS-primed murine BMDMs were stimulated for 6 h with nano-SiO 2 or nano-TiO 2 in the presence of the PLC- β inhibitor U73122 ( a ), the chelating molecule 2-APB, which blocks iCa 2+ increase ( b ), the ADCY activator forskolin ( c ) or the ADCY inhibitor SQ22536 ( d ). IL-1 β production was measured by ELISA. 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: Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay