Role of PDE4 in superoxide anion generation through p44/42MAPK regulation: a cAMP and a PKA-independent mechanism.
We investigated the generation of reactive oxygen species (ROS) from bronchoalveolar lavage (BAL) cells of either control or LPS-exposed rats and the effects of PDE4 inhibitors on ROS production. The PDE4 inhibitors, rolipram and Ariflo (cilomilast, SB 207499) dose-dependently (0.1-10 microm) inhibited fMLP-induced superoxide anion (O(2)(*-)) production (IC(50)s: 0.03 and 0.55 microm, respectively) in BAL cells of Wistar rats collected 3 h after an LPS-aerosol (200 micrograms ml(-1), 1 h). These BAL contained 85-95% neutrophils (BAL cells enriched in neutrophils). In contrast, BAL cells collected at the end of the challenge contained only macrophages and in these conditions, rolipram and Ariflo (0.1-10 microm) could only inhibit 25 and 45% of fMLP-induced O(2)(*-) release, respectively. We also observed that the inhibition of p44/42(MAPK) by PD98059 (1-10 microm) increased O(2)(*-) release by BAL cells enriched in neutrophils, but not by macrophages, and prevented the inhibition of O(2)(*-) production induced by PDE4 inhibitors. Western blot analysis showed that PDE4 inhibitors strongly activated p44/42(MAPK) in BAL cells enriched in neutrophils but not in macrophages. And in these cells, PDE4 and p44/42(MAPK) were co-immunoprecipitated by a polyclonal anti-PDE4 antibody. The following cell permeable-cAMP analogues, dbcAMP (10 microm-1 mm), 8-CPT-cAMP (1 mm) and 8-pMeOPT-2'-O-Me-cAMP (0.5 mm), could not reduce fMLP-induced O(2)(*-) production and both PKA inhibitors, PKA inhibitor 14-22 amide myristoylated (50 nm-1 microm) and H-89 (100 nm-1 microm), did not affect the decrease of O(2)(*-) release induced by PDE4 inhibitors in BAL cells enriched in neutrophils. These data suggest that PDE4 inhibitors decreased fMLP-induced O(2)(*-) release in BAL cells enriched in neutrophils but not in macrophages, through p44/42(MAPK) activation by a cAMP- and a PKA-independent mechanism.
Pfizer Global R&D, Fresnes Laboratories, Department of Biology, 3-9 rue de la Loge, 94265 Fresnes, France. cjacob@itsa.ucsf.edu
Claire
C
Jacob
Corinne
C
Szilagyi
Janet M
JM
Allen
Claude
C
Bertrand
Vincent
V
Lagente
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000502:physiology*; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D003994:Bucladesine / Q000494:pharmacology; D050882:CREB-Binding Protein; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D009570:Nitriles; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D020889:Rolipram / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D003509:Cyclohexanecarboxylic Acids; D005419:Flavonoids; D007093:Imidazoles; D008070:Lipopolysaccharides; D009570:Nitriles; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D017382:Reactive Oxygen Species; D015534:Trans-Activators; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; C433247:Cilomilast; D000242:Cyclic AMP; D050882:CREB-Binding Protein; C494365:Crebbp protein, rat; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one