Short Term Feedback Regulation of cAMP in FRTL-5 Thyroid Cells

Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R p)-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8-(4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt2cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control-transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal. Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R p)-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8-(4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt2cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control-transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal. protein kinase A phosphodiesterase follicle stimulating hormone human choriogondatropin phosphate-buffered saline radioimmunoassay 8-(4-chlorophenylthio)adenosine-cAMP dibutyryl cAMP Hormone or neurotransmitter signaling is mediated by transient fluctuations in intracellular cAMP within a very narrow range of concentrations (1.Soderling T.R. Corbin J.D. Park C.R. J. Biol. Chem. 1973; 248: 1822-1829Abstract Full Text PDF PubMed Google Scholar). Maximal biological effects are elicited with only 2–3-fold changes in intracellular cAMP levels, while the cell potential for cAMP production is usually much larger. In addition, the increase in intracellular cAMP is transient despite the continuous presence of the extracellular stimulus (2.Su Y.F. Cubeddu L. Perkins J.P. J. Cyclic Nucleotide Res. 1976; 2: 257-270PubMed Google Scholar, 3.Barber R. Clark R.B. Kelly L.A. Butcher R.W. Adv. Cyclic Nucleotide Res. 1978; 9: 507-516PubMed Google Scholar). This limited and short-lived nature of the activating signal is an essential feature of hormone or neurotransmitter action. This is necessary to decrease the intrinsic “noise” in the signaling mechanism, to allow iterative signaling, and to prevent excessive stimulation. The rapid and transient changes in cAMP concentrations are the result of changes in both synthesis and degradation of the second messenger cAMP, involving steps at the receptor as well as at a postreceptor level (4.Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar, 5.Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (402) Google Scholar). Following the activation of Gs and adenylyl cyclase, receptor phosphorylation causes an uncoupling from Gs, and therefore a decrease in cAMP synthesis. For the β2-adrenergic receptor, β2-adrenergic receptor kinases phosphorylate only agonist-occupied active receptors and enhance the affinity of the receptors for the inhibitor protein β-arrestin (6.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar). Binding of β-arrestin to the phosphorylated receptors inhibits the receptor-Gs interaction, thereby inducing the uncoupled or desensitized state of the receptors (7.Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar). Several kinases have also been implicated in the phosphorylation of the glycoprotein receptors including the GRK kinases, PKA1 and PKC. While PKA efficiently phosphorylates and uncouples the β-adrenergic receptor (5.Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (402) Google Scholar), the involvement of this kinase in the glycoprotein receptor phosphorylation is less clear (8.Ascoli M. Biochem. Pharmacol. 1996; 52: 1647-1655Crossref PubMed Scopus (20) Google Scholar). In addition to receptor uncoupling from G protein and cyclase, rapid modulation of phosphodiesterases and of cAMP degradation plays an essential role in the transient accumulation of cyclic nucleotides (9.Conti M. Nemoz G. Sette C. Vicini E. Endocr. Rev. 1995; 16: 370-389Crossref PubMed Scopus (317) Google Scholar). This concept was initially inferred by the use of xanthine inhibitors of PDEs and by measuring the decay of the cAMP signal in intact cells (3.Barber R. Clark R.B. Kelly L.A. Butcher R.W. Adv. Cyclic Nucleotide Res. 1978; 9: 507-516PubMed Google Scholar). The use of cAMP analogs has confirmed that cAMP-dependent PKA activation in the cell causes an activation of cAMP degradation (10.Corbin J.D. Gettys T.W. Blackmore P.F. Beebe S.J. Francis S.H. Glass D.B. Redmon J.B. Sheorain V.S. Landiss L.R. Methods Enzymol. 1988; 159: 74-82Crossref PubMed Scopus (27) Google Scholar, 11.Gettys T.W. Blackmore P.F. Redmon J.B. Beebe S.J. Corbin J.D. J. Biol. Chem. 1987; 262: 333-339Abstract Full Text PDF PubMed Google Scholar, 12.Corbin J.D. Beebe S.J. Blackmore P.F. J. Biol. Chem. 1985; 260: 8731-8735Abstract Full Text PDF PubMed Google Scholar). The impact and physiological significance of this rapid feedback regulation is unclear. Of the many PDEs expressed in the cell, two isoforms are activated by an increase in cAMP. In platelets (13.Macphee C.H. Reifsnyder D.H. Moore T.A. Beavo J.A. J. Cyclic Nucleotide Protein Phosphorylation Res. 1986; 11: 487-496PubMed Google Scholar, 14.Grant P.G. Mannarino A.F. Colman R.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9071-9075Crossref PubMed Scopus (61) Google Scholar) and adipocytes (15.Gettys T.W. Vine A.J. Simonds M.F. Corbin J.D. J. Biol. Chem. 1988; 263: 10359-10363Abstract Full Text PDF PubMed Google Scholar, 16.Manganiello V.C. Smith C.J. Newman A.H. Rice K. Degerman E. Belfrage P. J. Cyclic Nucleotide Protein Phosphorylation Res. 1986; 11: 497-511PubMed Google Scholar) a type 3 PDE is activated by a PKA-dependent phosphorylation lowering cAMP levels. Recently, a distinct PDE isoenzyme, a member of the PDE4 family, has been implicated in this feedback regulation (17.Sette C. Iona S. Conti M. J. Biol. Chem. 1994; 269: 9245-9252Abstract Full Text PDF PubMed Google Scholar). In thyroid cells, TSH causes a PKA-mediated phosphorylation and activation of a PDE4D3 variant. This conclusion is supported by studies involving PDE4-specific inhibitors and immunoprecipitation with PDE4-selective antibodies (17.Sette C. Iona S. Conti M. J. Biol. Chem. 1994; 269: 9245-9252Abstract Full Text PDF PubMed Google Scholar), and is consistent with cell-free phosphorylation and activation of the recombinant PDE4D3 enzymes (18.Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar). The site of PKA phosphorylation of PDE4D3 has been mapped to the amino terminus of PDE4D3 where Ser13 and Ser54 are the predominant phosphorylation sites. Only Ser54phosphorylation, however, is essential for activation of cAMP hydrolysis (18.Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar). Similar PDE4 activation has been described in myoblasts (19.Ball E.H. Seth P.K. Sanwal B.D. J. Biol. Chem. 1980; 255: 2962-2968Abstract Full Text PDF PubMed Google Scholar), aortic smooth muscle cells (20.Ekholm D. Belfrage P. Manganiello V. Degerman E. Biochim. Biophys. Acta. 1997; 1356: 64-70Crossref PubMed Scopus (25) Google Scholar), and osteoclasts (21.Ahlstrom M. Lamberg-Allardt C. J. Bone Miner. Res. 1997; 12: 172-178Crossref PubMed Scopus (34) Google Scholar), indicating the ubiquitous nature of this feedback regulation. While the impact of overall PDE4 activity on cAMP levels during cell stimulation can be assessed using PDE4-selective inhibitors, the exact role of PDE4D activation during hormonal stimulation has not been explored. Here, we have taken advantage of different strategies to distinguish between the effect of basal and activated PDE on cAMP accumulation and determine how the increase in PDE4D activity affects the TSH-dependent responses of FRTL-5 cells. We demonstrate that PDE4D3 activation has a major impact on the cAMP response by controlling the intensity of the cAMP signal. Coon's modified Ham's F-12 medium (Coon's F-12), bovine TSH, bovine insulin, human transferrin for culture, andCrotalus atrox snake venom were purchased from Sigma; bovine TSH, bovine FSH, and hCG for stimulation were obtained from the National Hormone and Pituitary Agency of the NIDDK, National Institutes of Health; Pansorbin cells from Calbiochem (La Jolla, CA); Immobilon membrane from Millipore Corp. (Bedford, from from cAMP analogs from from was by were the from FRTL-5 cells a of thyroid cells by S. L.A. Proc. Natl. Acad. Sci. U. S. A. 1980; PubMed Scopus Google Scholar), were by of NIDDK, National Institutes of and the were in Coon's F-12 medium with and a of including TSH and transferrin Leydig tumor cells M. PubMed Scopus Google Scholar) were in modified to and FRTL-5 cells and MA-10 cells were in at in an of and in a cell were from a M. L. R. M. PubMed Scopus (61) Google Scholar), and cells were on in a essential medium with amino and Incubation was at in a of and in a MA-10 cells were in at a of and MA-10 cells were transfected with PDE or as control C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar). was during a with as described by and J. PubMed Google Scholar). of The cells were with for at at the of the with the Following the cells were with and in medium for The medium was with medium with The cells were in this medium for with were by and with a and were to of a The were by to and These were in the medium and were or as were and for activity or for cAMP accumulation FRTL-5 cells were in in Coon's F-12 and cell were with and were for an with Coon's F-12 medium bovine to cell The medium was the MA-10 cells were in medium The medium was on and the were on the of the cells were with medium modified to of bovine the of the cell were with in and PDE activity was using cAMP as to the of and PubMed Scopus Google Scholar) as M. L. R. M. PubMed Scopus (61) Google Scholar). were in a of of including cAMP, bovine and In to of were to the at for min, the was by an of by for at of C. atrox snake venom was and the was at for min. The were by on and the of was by H89 is a that has been to be the and inhibitor of PKA in this of M. R. Methods Enzymol. PubMed Scopus Google Scholar). is an inactive to H89 was as a control for the effects of recombinant PDE4D3 activity in a in a cell-free H89 was less with of and a of both blocked of the PDE The FRTL-5 cell in were using cells to the of with C.H. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; PubMed Scopus Google Scholar). were with Pansorbin and an S. M. Sette C. M. Conti M. Pharmacol. PubMed Scopus Google Scholar). The PDE was from the by with in at were in and The were for 5 and were on an The were to an Immobilon and sites were blocked by the membrane in bovine in The the membrane was in a the of PDE4D3 S. M. Sette C. M. Conti M. Pharmacol. PubMed Scopus Google Scholar), in for for with changes of and for with in in for at antibodies were using a and after to FRTL-5 cells were in in Coon's F-12 and cell were with and were for an with Coon's F-12 medium bovine to cell The medium was the MA-10 cells were in medium The medium was on and the were on the of the the cells were with medium modified to of bovine cells were in medium The medium was on 3 and the were on the of the the cells were with the of the treatment in was were and to were by for at at in the was using at and the was with cAMP was by L.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) after of the and G. J. Cyclic Nucleotide Res. Google Scholar). for cell stimulation was by on a (10.Corbin J.D. Gettys T.W. Blackmore P.F. Beebe S.J. Francis S.H. Glass D.B. Redmon J.B. Sheorain V.S. Landiss L.R. Methods Enzymol. 1988; 159: 74-82Crossref PubMed Scopus (27) Google Scholar). were with for at and was by the addition of in were at at in the was using at and the was with The was at to a in and were J.D. Beebe S.J. Blackmore P.F. J. Biol. Chem. 1985; 260: 8731-8735Abstract Full Text PDF PubMed Google Scholar). to cAMP were and with and cAMP was by of from the was Protein was by the of Biochem. 1976; PubMed Scopus Google Scholar) or R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) using bovine or as a The PDE activity expressed in FRTL-5 cells is by TSH (17.Sette C. Iona S. Conti M. J. Biol. Chem. 1994; 269: 9245-9252Abstract Full Text PDF PubMed Google Scholar). Under the the increase in PDE activity was maximal within 5 min, and the activity for at We have that this PDE activation is with phosphorylation and a shift in mobility of the PDE4D3 protein (18.Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar). A showed that a with an of was in cells, and with TSH caused the of an of The of the was with a decrease in the intensity of the PDE4D3 phosphorylated for at after stimulation and the shift in mobility was consistent with the increase in the role of PKA on activation of FRTL-5 cells were for in the or presence of either H89, a or (R p)-cAMPS, a cAMP the of this cells were with TSH for 5 min, and PDE activity was in the cell TSH stimulation of PDE was blocked by either H89 or (R with H89 the PDE activity at of PKA inhibitors on the PDE between these two is between these two is FRTL-5 cells were in the or presence of different PKA inhibitors H89, for the of the cells were for 5 with in and the PDE activity in the cell was using as The between these two is in a FRTL-5 cells were in the or presence of different PKA inhibitors H89, for the of the cells were for 5 with in and the PDE activity in the cell was using as determine the PKA is with a in mobility of the PDE4D3 expressed in FRTL-5 cells, was on from cells and with TSH and different PKA 3 that the shift in the mobility of the was blocked by H89, and to a by while the isomer effect The shift was blocked by a with (R not these data that phosphorylation and activation of the PDE4D3 protein was by PKA activation of a PDE is by H89, of PKA was to determine the impact of PDE feedback activation on cAMP levels in FRTL-5 cells. FRTL-5 were with H89 or for to and during the TSH and cAMP levels were H89 treatment potentiated the TSH-dependent cAMP but effect A showed that cAMP accumulation was by H89 PKA as as 3 and that H89 to be after of the TSH-dependent cAMP effect of the PKA inhibitor FRTL-5 cells were in the or presence of H89 for the of the cells were with TSH. were at different from the hormone addition and intracellular cAMP levels were by the of A of the is The PDE4-specific inhibitor at a of a increase in cAMP accumulation induced by TSH, with major in the not with the PDE inhibitor the of cAMP to basal levels not was in with H89 and TSH, the H89 of cAMP accumulation was TSH H89 of of This that the H89 effect was not to changes in the of cAMP synthesis. and FRTL-5 cells have been compared provide with to hormone receptor activation of cells. While TSH a rapid activation of a PDE in FRTL-5 cells, not PDE activity in a in cells. This is to the that PDE4D3, the for PKA activation in FRTL-5 cells, is in cells C. Vicini E. Conti M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). cells were to determine H89 treatment potentiated the cAMP accumulation in the of a PDE activation. that H89 not the stimulation of cAMP in the cells during this or a decrease in cAMP accumulation was with H89 after and This that H89 was in intracellular cAMP when PDE4D3 activation was to the feedback a activation of PKA the adenylyl a decrease in cAMP in the This was by FRTL-5 cells with cAMP analogs that can be from the endogenous cAMP. this has been in advantage of cAMP analogs that PKA S.J. Corbin J.D. The Scholar). can be from cAMP on a (10.Corbin J.D. Gettys T.W. Blackmore P.F. Beebe S.J. Francis S.H. Glass D.B. Redmon J.B. Sheorain V.S. Landiss L.R. Methods Enzymol. 1988; 159: 74-82Crossref PubMed Scopus (27) Google Scholar). In a FRTL-5 cells were or with different concentrations of for min, PDE activity was with data obtained with TSH and dibutyryl cAMP, treatment caused an increase in PDE activity endogenous cAMP was and after treatment of the FRTL-5 cells, a decrease in cAMP was The decrease was on the of the this the presence of a feedback in FRTL-5 cells cAMP and involving PKA phosphorylation of the impact of stimulation of PDE4D3, a where PDE4D3 is expressed in a hormone cell was A Leydig tumor cell S. M. Sette C. M. Conti M. Pharmacol. PubMed Scopus Google Scholar) not PDE4D or protein. These cells of is not activated by either in a or Conti M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). an that for wild type PDE4D3 or PDE4D3 mutant in two Ser13 and are with (18.Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar), cell these two were in the presence of cells were with hCG or and PDE activity was in the cell cells not changes in PDE activity after stimulation with either hCG or hCG or Bt2cAMP treatment PDE activity in cells transfected with PDE4D3 wild type Incubation with to hCG stimulation potentiated the PDE activation. In cells transfected with mutant PDE4D3, the basal PDE activity was that in control cells but the stimulation by hCG or Bt2cAMP was that of an of was expressed in the PDE4D3 and in the PDE4D3 mutant cell but not in the control cell Bt2cAMP treatment caused a shift in the mobility of the PDE4D3 wild type protein but not of the mutant PDE4D3 not the PDE activation can be to a cell by the of In to determine the impact of PDE4D3 activation on cAMP the transfected cells were with hCG for and the intracellular cAMP levels were The cAMP accumulation was in cells transfected with wild type PDE4D3 while cells transfected with PDE4D3 mutant the of control cells. treatment with the of the cells, and in the presence of the PDE inhibitors, cell were to hCG These data demonstrate that activation of PDE4D3 in a cell has a major impact on cAMP the was by Rolipram, we also that the in response is to the transfected PDE and not to mechanisms induced during the of the cell The studies demonstrate the presence of a feedback that cAMP levels in FRTL-5 thyroid cells and phosphorylation of a Following TSH receptor and activation of adenylyl cyclase, an increase in intracellular cAMP PKA that in phosphorylates and of PDE4D3 and the increase in the of degradation the cAMP signal. The presence of this feedback is by activating or PKA in FRTL-5 cells as well as by a where cAMP levels are reduced when a PDE4D3 is In addition, this feedback regulation is not active in cells where PDE4D3 is not expressed or a phosphorylation mutant is In was to the effect of the PDE activation from the basal PDE activity in the H89 has been to PKA-dependent M. R. Methods Enzymol. PubMed Scopus Google Scholar), has been to also signaling steps the PKA we have that in a cell-free H89 inhibits PDE4D activity with an of This effect on PDE however, be the of the increase in cAMP in FRTL-5 cells to the of only of PDE activity in a cell-free and concentrations are when the intact cell is to this H85, is H89 in the PDE effect on cAMP the that the PDE is the of the G. J. C.J. J. Biol. Chem. 1995; PubMed Scopus Google Scholar) have that PKA phosphorylates type adenylyl cyclase, the major expressed in and thereby inhibits This is in the presence of a PKA that PKA-mediated phosphorylation and of adenylyl a for of the G receptor this is in FRTL-5 cells is unclear. data that H89 has effect in the presence of that of PKA affects the of cAMP degradation but not the of synthesis by adenylyl is also that H89 the TSH-dependent increase in cAMP by a PKA-mediated phosphorylation of the TSH receptor This is a described for the β-adrenergic receptor where a PKA-mediated phosphorylation causes uncoupling of the receptor from is at clear evidence that PKA phosphorylates the TSH or glycoprotein hormone receptors as for the β-adrenergic Several have that PKA-dependent phosphorylation not an role in uncoupling glycoprotein hormone receptors M. Biochem. Pharmacol. 1996; 52: 1647-1655Crossref PubMed Scopus Google Scholar). these that an cAMP accumulation is caused by a of steps at the level of the the conclusion that H89 causes an increase in cAMP by PDE activation is consistent with the in cells. the receptor is to the TSH receptor, H89 not cAMP accumulation in these cells where the feedback activation of a PDE is not In but not treatment of cells with H89 caused a decrease in cAMP this was only after of this be to the PKA of cAMP accumulation that PDE activation causes a of the for cAMP levels to a (3.Barber R. Clark R.B. Kelly L.A. Butcher R.W. Adv. Cyclic Nucleotide Res. 1978; 9: 507-516PubMed Google Scholar). PDE a in the of maximal cAMP the that be to cAMP after addition of in studies with either H89 or Rolipram, we that the to a was not after PDE4D3 activation. the major was a in the level of cAMP in the the in cAMP basal levels was not whereas is that PDE activation to the cAMP signal. A major impact of cAMP from the cell is at these cAMP in in the extracellular is less of the intracellular cAMP. the of these we that the feedback involving PDE4D3 activation the intensity of the cAMP while impact on the of cAMP accumulation or the of the stimulus is less PDEs adenylyl a role in the of cAMP to basal levels. The effect of on the cyclic was of the effect of the PDE activation with This is to the that both the transient PDE4D activation as well as the basal activity of in the PDE4D3 has activity in a cell-free when in the PKA phosphorylation sites (18.Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar), is a is active in the intact are to this In we have that PDE4D3 phosphorylation and activation is a cAMP This is activated by cAMP, and with the as adenylyl activation. that this PDE regulation the of cAMP synthesis and that affects the maximal cAMP accumulation the of cAMP from the this regulation a to cAMP within a narrow range of also be a necessary to the in cAMP signaling and to the of cAMP from the site of synthesis. The physiological of this PDE4D regulation is by the of the PDE4D cells in a PDE4D a reduced to hormone that the PDE4 feedback the cell from W.P. A.J. Conti M. Proc. Natl. Acad. Sci. U. S. A. Scholar). We are to for on the