SALBUTAMOL UP-REGULATES PDE4 ACTIVITY AND INDUCES A HETEROLOGOUS DESENSITIZATION OF U937 CELLS TO PROSTAGLANDIN E(2) - IMPLICATIONS FOR THETHERAPEUTIC USE OF BETA-ADRENOCEPTOR AGONISTS

Previous studies with U937 cells, a human monocyte cell line, have sho wn that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) i s increased by agents that elevate cyclic AMP content, The present exp eriments were conducted to determine 1) whether an increase in PDE4 st eady-state message and/or protein accompanies the up-regulation of PDE 4 activity and 2) whether the up regulation changes the functional res ponses 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 mu M salbutamol, a beta-adrenoceptor agonist, and 30 mu M rolipram, a PDE4 inhibitor, Cells were washed extensively to remove drugs and us ed immediately in various experimental protocols, Reverse transcriptas e-polymerase chain reactions conducted with primers specific for the f our PDE4 subtypes suggested that pretreatment with salbutamol and roli pram increased steady-state mRNA levels of PDE4A and PDE4B, but not PD E4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodi es, one directed against human recombinant PDE4A and PDE4D and a secon d directed against human recombinant PDE4B, revealed bands of immunore activity corresponding to similar to 125 kDa (PDE4A) and similar to 70 MDa (PDE4B), respectively, that increased in intensity after cells we re treated with salbutamol and rolipram, As demonstrated in both time course and concentration-response studies with prostaglandin E(2) (PGE (2)), an agent that activates adenylyl cyclase by a non-beta-adrenocep tor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up regulated. The difference in PGE(2)-stimulated cAMP accumulation be tween control and PDE4 up-re gulated cells was greatly reduced in the presence of rolipram, consist ent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, upregulation of PD E4 markedly decreased the ability of PGE(2) to inhibit LTD(4)-induced Ca2+ mobilization in intact cells, A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering bet a-adrenoceptor agonists could exacerbate inflammatory processes by dec reasing the activity of endogenous antiinflammatory agents such as PGE (2).