Comparative Analyses of Cyclic Adenosine 3́:5́-monophosphate Phosphodiesterases of Human Peripheral Blood Monocytes and Cultured P388D₁ Cells

Human monocytes (95% purity) were isolated from defibrinated venous blood by centrifugation through Ficoll-Hypaque followed by adherence to polystyrene dishes. Freshly isolated monocytes or those cultured for 6 days contained a soluble high-affinity cyclic adenosine 3́:5́-monophosphate (cyclic AMP) phosphodiesterase (K_m, 1.1 ± 0.2 μM) that resembled the enzyme from human peripheral blood lymphocytes (HPBL), which was described previously. Other similarities include: (a) lack of activity in particulate fractions of the cell; (b) linear Lineweaver-Burk plots of cyclic AMP hydrolysis; and (c) phosphodiesterase activities sedimenting at 3.5 to 4.0S and 5.5 to 6.0S as determined by centrifugation on linear sucrose gradients. The monocyte enzyme had a higher maximum velocity (9.0 ± 2.4 pmol/min/10⁶ cells) than that from HPBL. Like that from HPBL, the activity of the 3.5 to 4.0S enzyme form of monocytes was greatly elevated after incubation of sonicates for 24 hr at 4° or after culture of the cells with 1-methyl-3-isobutylxanthine for 24 hr. Unlike HPBL, activation of the phosphodiesterase by 1-methyl-3-isobutylxanthine was blocked completely by 7 μM cycloheximide. The high-affinity cyclic AMP phosphodiesterase activity of freshly isolated monocytes was not affected by phagocytosis of styrene beads, by killed Staphylococcus aureus, or by culture with insulin (4 or 200 nm). No cyclic guanosine 3́:5́-monophosphate phosphodiesterase activity was detectable in monocyte sonicates. The “macrophage-like” P388D₁ cell line contained cyclic AMP phosphodiesterase activity which exhibited nonlinear kinetic plots and maximum velocities higher than that from HPBL or monocytes. Culture of P388D₁ cells for 24 hr with 1-methyl-3-isobutylxanthine and/or insulin did not affect cyclic AMP phosphodiesterase activity. These differences from monocytes further emphasize that the P388Di cell line is not “macrophage-like” in relation to cyclic AMP metabolism.