selective pde4 inhibitor rolipram  (Tocris)

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: P2Y 11 receptor‐driven release of soluble TNF receptors depends on intracellular cAMP and on TACE/ADAM17: synergistic enhancement by IL‐1α and IL‐1ß (a) M2 macrophages were treated with ATPγS in the presence or absence of increasing concentrations of the phosphodiesterase (PDE) inhibitors IBMX (nonselective) or rolipram (PDE4‐selective). The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. sTNFR2 was measured in cell culture supernatants. (b) M2 macrophages were treated with ATPγS in the presence or absence of increasing concentrations of the TACE/ADAM17 inhibitors TAPI‐1 and TAPI‐2. The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. sTNFR2 was measured in cell culture supernatants. (c) M2 macrophages were treated for 24 h with increasing doses of IL‐1α or IL‐1ß either alone or in combination with the P2Y 11 receptor agonist ATPγS (10 μM). The antagonist NF340 (10 μM) was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. For all graphs, data shown are means ± SEM from five independent donors. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: Additional reagents used in this study are as follows: P2X1 receptor antagonist NF449 (10 μM) (Tocris), adenylyl cyclase inhibitor SQ22536 (5 to 20 μM) (Tocris), Gq/11 inhibitor YM‐254890 (0.1 to 0.5 μM) (Adipogen Life Sciences, Liestal, Switzerland), recombinant IL‐1α and IL‐1ß (50 to 1,000 pg·ml −1 ) (R&D, Bio‐Techne), recombinant IL‐1 receptor antagonist (IRAP ; 0.05 to 0.2 μg·ml −1 ) (R&D, Bio‐techne); nonselective phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX ) (20 to 200 μM) (Sigma‐Aldrich); PDE4‐ selective inhibitor rolipram (2 to 10 μM) (Sigma‐Aldrich), the TACE/ADAM17 inhibitors TAPI‐1 and TAPI‐2 (10 to 50 μM) (Tocris); lipopolysaccharide (LPS) from Salmonella abortus equi (10 pg·ml−1 to 10 ng·ml −1 ) (Sigma‐Aldrich) was used to activate M2 macrophages via TLR4.

Techniques: Cell Culture

Journal: Neuron

Article Title: Precisely-timed nicotinic activation drives SST inhibition in neocortical circuits

doi: 10.1016/j.neuron.2018.01.037

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Pharmacology The following agents were used for bath-application in acute brain slice experiments: forskolin, cell-permeable activator of adenylyl cyclase (50 μM, Tocris), selective cAMP-specific phosphodiesterase (PDE4) inhibitor rolipram (0.1 μM, Sigma), the noradrenergic agonist norepinephrine (50 μM, Sigma), the serotonin receptor agonist serotonin (10 μM, Tocris), the dopamine receptor agonist CY 208–243 (50 μM, Tocris), P2 receptor agonist adenosine (100 μM, Sigma), the CB1 receptor antagonist AM 251 (1 μM, Tocris), the group I metabotropic glutamate receptor agonist DHPG (10 μM, Sigma).

Techniques: Plasmid Preparation, Recombinant, Software

Journal:

Article Title: Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans

doi: 10.1152/ajpheart.01226.2008

Figure Lengend Snippet: Model of PDE activity localized with the β2-adrenergic receptor (β2AR) and PGI2 receptor (IPR) in rabbit and human erythrocytes. Activation of the IPR leads to generation of cAMP in a restricted cAMP pool that is hydrolyzed by PDE3. In contrast, activation of the β2AR leads to generation of a distinct cAMP pool that is regulated by the activities of both PDE2 and PDE4. AC, adenylyl cyclase; Gs, heterotrimeric G protein.

Article Snippet: The inhibitors studied were vinpocetine, a selective PDE1 inhibitor (Sigma- Aldrich); 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor (Sigma-Aldrich); rolipram, a selective PDE4 inhibitor (Tocris); erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), a selective PDE2 inhibitor (Biomol); and cilostazol, a selective PDE3 inhibitor (Sigma-Aldrich).

Techniques: Activity Assay, Activation Assay

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: (a) Percentage of reactive oxygen species (ROS) production in bronchoalveolar lavages (containing 90% neutrophils) of LPS-treated rats 10 min after fMLP (2 μM) addition in the presence of MAPK inhibitors, PKA inhibitor or PKC inhibitor and/or PDE4 inhibitors (C+=positive control, R=rolipram, A=Ariflo, SB=SB203580, PD=PD98059, PKAi=0.05 μM protein kinase A inhibitor 14–22 amide myristoylated, PKCi=0.4 μM protein kinase C inhibitor, W=0.05 μM wortmannin). This figure represents the results of 15 experiments. (b) Percentage of reactive oxygen species (ROS) production in bronchoalveolar lavages (containing 90% neutrophils) of LPS-treated rats 10 min after fMLP (2 μM) addition in the presence of cAMP analogs (DbcAMP=1 mM, 8-pMeOPT=1 mM 8-pMeOPT-2′-O-Me-cAMP), PKA inhibitors (14–22=0.1–1 μM protein kinase A inhibitor 14–22 amide myristoylated, H-89=0.1–1 μM) and/or PDE4 inhibitors (R=10 μM rolipram, A=10 μM Ariflo). This figure represents the results of five experiments. Statistical analysis for (a) and (b): the effects of each compound were compared to a theoretical mean of 100% representing the positive control (*P<0.01, **P<0.001, ***P<0.0001), and the effects of rolipram or Ariflo in the presence of MAPK, PKA, PKC inhibitors or cAMP analogs were compared to the effects of rolipram or Ariflo alone (†P<0.01, ††P<0.001, †††P<0.0001), using the Holm's multiple test procedure.

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Positive Control

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: Percentage of reactive oxygen species (ROS) production in bronchoalveolar lavages (containing 98% macrophages) of LPS-treated rats at the activation peak induced by 2 μM fMLP in the presence of PDE4 inhibitors or MAPK inhibitors (C+=positive control, W=0.05 nM wortmannin). Statistical analysis: the effects of each compound were compared to a theoretical mean of 100% representing the positive control, using the Holm's multiple test procedure (*P<0.01, **P<0.001, ***P<0.0001).

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Activation Assay, Positive Control

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: (a) Western blot of phospho-CREB in BAL neutrophils of LPS-treated rats 30 min after 1 mM DbcAMP addition in the presence of PKA inhibitors (14–22=1 μM protein kinase A inhibitor 14–22 amide myristoylated, H-89=1 μM). (b) Western blot of phospho-CREB in BAL neutrophils of LPS-treated rats 30 min after 10 μM forskolin addition in the presence of PDE4 inhibitors (rolipram=0.1–10 μM, Ariflo=0.1–10 μM).

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Western Blot

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: (a) Western blot of phosphorylated MKK3 and percentage of phosphorylated MKK3 evaluated by densitometry (OD mm−2) of the blots in bronchoalveolar lavages of LPS-treated rats 10 min after 2 μM fMLP addition in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, W=0.05 nM wortmannin). (b) Western blot of phosphorylated MKK6 and percentage of phosphorylated MKK6 evaluated by densitometry (OD mm−2) of the blots in bronchoalveolar lavages of LPS-treated rats 10 min after 2 μM fMLP addition in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, W=0.05 nM wortmannin).

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Western Blot, Negative Control, Positive Control

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: (a) Western blot of phosphorylated p44/42MAPK and percentage of phosphorylated p44/42MAPK evaluated by densitometry (OD mm−2) of the blots in BAL neutrophils of LPS-treated rats 10 min after 2 μM fMLP addition in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, W=0.05 nM wortmannin). (b) Western blot of phosphorylated p44/42MAPK and percentage of phosphorylated p44/42MAPK evaluated by densitometry (OD mm−2) of the blots in BAL macrophages of LPS-treated rats at the activation peak induced by 2 μM fMLP in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, W=0.05 nM wortmannin).

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Western Blot, Negative Control, Positive Control, Activation Assay

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: Western blot of phosphorylated p38MAPK and percentage of phosphorylated p38MAPK evaluated by densitometry (OD mm−2) of the blots in bronchoalveolar lavages of LPS-treated rats 10 min after 2 μM fMLP addition in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, SB=SB203580, PD=PD98059, W=0.05 nM wortmannin).

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Western Blot, Negative Control, Positive Control

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: (a) Western blot of phosphorylated c-Raf and Percentage of phosphorylated c-Raf evaluated by densitometry (OD mm−2) of the blots in bronchoalveolar lavages of LPS-treated rats 10 min after 2 μM fMLP addition in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, W=0.05 nM wortmannin). (b) Western blot of phosphorylated MEK1/2 and percentage of phosphorylated MEK1/2 evaluated by densitometry (OD mm−2) of the blots in bronchoalveolar lavages of LPS-treated rats 10 min after 2 μM fMLP addition in the presence of PDE4 inhibitors or MAPK inhibitors (C−=negative control, C+=positive control, PD=PD98059, W=0.05 nM wortmannin).

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Western Blot, Negative Control, Positive Control

Journal:

Article Title: Role of PDE4 in superoxide anion generation through p44/42 MAPK regulation: a cAMP and a PKA-independent mechanism

doi: 10.1038/sj.bjp.0705927

Figure Lengend Snippet: Western blot of p44/42MAPK and phospho-p44/42MAPK co-immunoprecipitated with PDE4 by the polyclonal anti-rat PDE4 antibody K116 in lysates of neutrophils from LPS-treated rat bronchoalveolar lavages (C−=control cells, C+=cells activated with fMLP (2 μM)) and re-blot of the membrane after stripping with K116.

Article Snippet: The selective PDE4 inhibitors Rolipram and Ariflo (cilomilast) were synthesized in our Department of Chemistry (Pfizer Global R&D, Fresnes Laboratories, France).

Techniques: Western Blot, Immunoprecipitation, Control, Membrane, Stripping Membranes