Salbutamol Up-regulates PDE4 Activity and Induces a Heterologous Desensitization of U937 Cells to Prostaglandin E2

Abstract

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 M salbutamol, a ␤-adrenoceptor agonist, and 30 M 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 ϳ125 kDa (PDE4A) and ϳ70 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 E 2 (PGE 2 ), an agent that activates adenylyl cyclase by a non-␤-adrenoceptor-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 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, upregulation of PDE4 markedly decreased the ability of PGE 2 to inhibit LTD 4 -induced Ca 2؉ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering ␤-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous antiinflammatory agents such as PGE 2 .

Identification of cyclic AMP‐phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells

“…The anti-PDE4 K116 also recognized a band of 120 kDa, which is not detected by M3S1, indicating that it is not a PDE4D isoform. The molecular weight of this PDE is similar to that predicted for PDE4A5 [22], and a band of similar size was recently detected in human U937 cells by using an anti-PDE4A antibody [37].…”

“…Previous studies on promonocytic cell lines pointed to the expression of PDE4D3 in unstimulated U937 cells [31,37,40], and to its absence in Mono Mac 6 cells [38]. The expression of other isoforms, namely PDE4D1, PDE4B2, and PDE4A5, was induced by cAMP-elevating agents in these cell lines [37,38,40].…”

“…Furthermore, it has been shown that, in the rat, the cAMP regulation of the activity of the long PDE4D3 variant occurs at the post-translational level, via a cAMP-dependent phosphorylation [27,29,30]. Studies with human U937 cells point to the absence of up-regulation of this variant by cAMP at the transcriptional level [37]. Moreover, in the same cell type, PDE4D3 undergoes a short-term regulation by PKA-phosphorylation [31], which strongly suggests that its regulation is similar in human and rat.…”

“…Previous studies on promonocytic cell lines pointed to the expression of PDE4D3 in unstimulated U937 cells [31,37,40], and to its absence in Mono Mac 6 cells [38]. The expression of other isoforms, namely PDE4D1, PDE4B2, and PDE4A5, was induced by cAMP-elevating agents in these cell lines [37,38,40]. Thus, there remains considerable uncertainty concerning the forms expressed in unstimulated normal human monocytes, and it is difficult to completely rule out the participation of this cell type to the isoenzyme expression pattern we have determined from blood mononuclear cells.…”

The human near‐term myometrial β3‐adrenoceptor but not the β2‐adrenoceptor is resistant to desensitisation after sustained agonist stimulation

“…There is now defined evidence that cAMP induces an upregulation of PDE4, accelerating the degradation of this cyclic nucleotide (Perry et al, 2002). Interestingly, previous studies on cellular models such as U937 monocytes (Torphy et al, 1995) and human neutrophils (Ortiz et al, 2000) indicated that cAMP-PDE4 activity was increased after a treatment by b 2 -agonists. In human myometrial cells, we demonstrated that compounds elevating intracellular cAMP level, such as 8-Br-cAMP or forskolin, were able to increase PDE4 activity (Mehats et al, 1999).…”