Action of rolipram on specific PDE4 cAMP phosphodiesterase isoforms and on the phosphorylation of cAMP-response-element-binding protein (CREB) and p38 mitogen-activated protein (MAP) kinase in U937 monocytic cells.
U937 monocytic cells are shown here to express a range of PDE4, cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes, PDE4A4, PDE4D5 and PDE4D3, plus the short isoenzyme, PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A, PDE4B and PDE4D activities were 0.63+/-0.09, 8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor, rolipram, inhibited immunopurified PDE4B and PDE4D activities similarly, with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast, rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis, it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes.
MacKenzie
S J
SJ
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson and Wolfson Buildings, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D019893:Ribosomal Protein S6 Kinases / Q000737:chemistry / Q000378:metabolism; D020889:Rolipram / Q000494:pharmacology*; D020123:Sirolimus / Q000494:pharmacology; D020298:U937 Cells; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases
D000730:Androstadienes; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D007371:Interferon-gamma; D019893:Ribosomal Protein S6 Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human; C517360:PDE4B protein, human; C517376:PDE4D protein, human; D020889:Rolipram; D020123:Sirolimus; D000077191:Wortmannin