Activation and selective inhibition of a cyclic AMP-specific phosphodiesterase, PDE-4D3.
Prostaglandin E2 produces a transient increase in the intracellular concentration of cAMP in a human promonocytic cell line (U937). The temporal pattern consists of a rapid increase followed by a gradual decline to a new steady state. The decline phase coincides with an increase in the activity of a high affinity form of cAMP phosphodiesterase (PDE). Immunoprecipitation with specific antibodies revealed that the activated enzyme is a variant of PDE-4D. To confirm this observation, three isoforms of human PDE-4 (A, B, and D) were cloned and expressed in Sf9 cells with recombinant baculovirus infection. The activity of only one of the isoforms (PDE-4D3) increased after incubation with the catalytic subunit of protein kinase A and Mg-ATP. Hydrolytic activity of human PDE-4D3 was dependent on Mg2+. Before phosphorylation, the concentration-response curve for Mg2+ was biphasic and ranged from 0.1 to 100 mM. Phosphorylation of PDE-4D3 by protein kinase A produced a monophasic Mg2+ response curve (0.5 Vmax = 0.2 mM). Phosphorylation of PDE-4D3 increased the sensitivity of the enzyme to inhibition by RS-25344 (approximately 100-fold) and RS-33793 (approximately 330-fold). Thus, phosphorylation of PDE-4D3 induces an apparent conformation change that increases maximum velocity and sensitivity to inhibition by some analogues of nitraquazone. These observations provide the basis for a novel pharmacological strategy that targets an activated form of PDE in human leukocytes. Selective PDE-4D3 inhibitors may have useful anti-inflammatory properties with fewer adverse side effects than other PDE-4 inhibitors.
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000242:Cyclic AMP / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D015232:Dinoprostone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011724:Pyridazines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011724:Pyridazines; D011799:Quinazolines; C096328:RS 25344; C096327:RS 33793; D011994:Recombinant Proteins; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D008274:Magnesium; D015232:Dinoprostone