Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists.
Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2.
Torphy
T J
TJ
Division of Pharmacological Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939, USA.
Zhou
H L
HL
Foley
J J
JJ
Sarau
H M
HM
Manning
C D
CD
Barnette
M S
MS
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000627:therapeutic use; D000420:Albuterol / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D015232:Dinoprostone / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020889:Rolipram; D015854:Up-Regulation / Q000187:drug effects
D000318:Adrenergic beta-Agonists; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D012333:RNA, Messenger; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram; D015232:Dinoprostone; D000420:Albuterol; D002118:Calcium