Stimulation of beta adrenoceptors in a human monocyte cell line (U937) up-regulates cyclic AMP-specific phosphodiesterase activity.
Experiments were conducted using undifferentiated U937 cells, a human monocytic cell line, to establish an in vitro model to examine the hormonal regulation of the cyclic AMP (cAMP)-specific phosphodiesterase (PDE IV). Standard chromatographic techniques, coupled with the use of inhibitors and activators that are selective for various phosphodiesterase (PDE) isozymes, were used to establish the PDE isozyme profile in supernatant fractions of U937 cells. When PDE activity was assessed using 1 microM [3H]cAMP as a substrate, 70 to 90% of the total U937 cell supernatant activity in the major peak eluting from anion-exchange columns was inhibited by 30 microM rolipram, a selective inhibitor of PDE IV. The remaining activity was nearly abolished by 10 microM siguazodan or 10 microM cyclic GMP (cGMP,) selective inhibitors of the cGMP-inhibited PDE. Kinetic analyses of the enzyme activity contained within this major peak of PDE activity revealed a cAMP Km = 3 microM and a rolipram Ki = 0.5 microM, values characteristic of PDE IV. Additional studies revealed the presence of a small amount of Ca++/calmodulin-stimulated PDE, but no cGMP-stimulated PDE or cGMP-specific PDE activity. In an effort to induce PDE activity in intact U937 cells by producing a sustained increase in cAMP content, cells were treated for 4 hr with salbutamol (1 microM), rolipram (30 microM) or a combination of both agents. The combination of salbutamol and rolipram produced a 2- to 3-fold increase in PDE activity in U937 cells; when used alone, rolipram was without effect whereas salbutamol induced an increase that was approximately one-half of that observed with the combination. Isozyme isolation and characterization revealed that the overall elevation of cellular PDE activity could be accounted for by a 2- to 3-fold increase in the Vmax of PDE IV with no change in its Km. The induction of PDE IV by salbutamol was: 1) concentration- and time-dependent; 2) detectable only after prolonged (2-4 hr) agonist exposure; 3) preceded by an increase in cAMP content and an activation of cAMP-dependent protein kinase; 4) mimicked by 8-bromo-cAMP and prostaglandin E2; 5) reversible within 3 hr of salbutamol removal; and 6) abolished by cycloheximide or actinomycin D. Collectively, these results indicate that the major PDE isozyme in the soluble fraction of U937 cells is PDE IV and that the activity of this enzyme is increased markedly in cells after prolonged exposure to agents that increase cAMP content.
Torphy
T J
TJ
Department of Inflammation & Respiratory Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania.
Zhou
H L
HL
Cieslinski
L B
LB
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D000420:Albuterol / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007951:Leukemia, Myeloid; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology*; D011494:Protein Kinases / Q000032:analysis; D011760:Pyrrolidinones / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D020889:Rolipram; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D011760:Pyrrolidinones; D011943:Receptors, Adrenergic, beta; D003609:Dactinomycin; D003513:Cycloheximide; D000242:Cyclic AMP; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram; D015232:Dinoprostone; D000420:Albuterol