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β-Adrenergic cAMP signals are predominantly regulated by phosphodiesterase type 4 in cultured adult rat aortic smooth muscle cells.

Zhai K, Hubert F, Nicolas V, Ji G, Fischmeister R, Leblais V - PLoS ONE (2012)

Bottom Line: Both β(1)- and β(2)-AR antagonists decreased the signal amplitude without affecting its kinetics.PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment.This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

View Article: PubMed Central - PubMed

Affiliation: Inserm UMR-S 769, LabEx LERMIT, Châtenay-Malabry, France.

ABSTRACT

Background: We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs).

Methodology/principal findings: The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3  =  PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β(1)- and β(2)-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β(1)- and β(2)-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation.

Conclusion/significance: Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

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Effect of PDE inhibitors on the peak amplitude and t1/2off of β-AR-induced cytosolic cAMP signal in RASMCs.Dynamic parameters (peak amplitude, A and t1/2off, B) obtained in Figure 5 are expressed in % of isoproterenol-induced response (Control). Data are mean±SEM of 8–13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 versus Control; $ P<0.05, $$ P<0.01 and $$$ P<0.001 versus IBMX; # P<0.05, ### P<0.001 as indicated.
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pone-0047826-g006: Effect of PDE inhibitors on the peak amplitude and t1/2off of β-AR-induced cytosolic cAMP signal in RASMCs.Dynamic parameters (peak amplitude, A and t1/2off, B) obtained in Figure 5 are expressed in % of isoproterenol-induced response (Control). Data are mean±SEM of 8–13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 versus Control; $ P<0.05, $$ P<0.01 and $$$ P<0.001 versus IBMX; # P<0.05, ### P<0.001 as indicated.

Mentions: To evaluate the functional contribution of the different PDE families in regulating the cAMP response to β-AR stimulation, RASMCs were incubated in the presence of PDE inhibitors during 3 min before Iso pulse. Application of PDE inhibitors alone, with the exception of BAY and BRL, induced a slight but significant increase in basal FRET ratio, which was maximal with 50 µM MIMX (the PDE1 inhibitor) and 100 µM IBMX (the non-selective PDE inhibitor) (Table2). 50 µM MIMX (Figure5A), 100 nM BAY (the PDE2 inhibitor, Figure5B) and 1 µM Cil (the PDE3 inhibitor, Figure5C) had no effect on the dynamics of the Iso-induced FRET signal. 50 µM BRL (the PDE7 inhibitor, Figure5E) had only minor effects on its kinetics (with a slight increase in the time to peak of 17%, n = 9, P<0.05), whereas 10 µM Ro (the PDE4 inhibitor, Figure5D) markedly increased its amplitude (by 52%, n = 13, P<0.01) and significantly delayed its onset and recovery phases. IBMX (Figure5I) also increased the amplitude of Iso response (by 76%, n = 9, P<0.05) and dramatically prolonged its duration. The IBMX effect on the decay phase was significantly higher than that induced by Ro (t1/2off increased by 99% and 770% with Ro and IBMX, respectively, P<0.001; Figure6B). This indicates that, in RASMCs, PDE4 is an essential regulator of β-AR-elicited cytosolic cAMP signals, but that other PDEs might also contribute to this regulation.


β-Adrenergic cAMP signals are predominantly regulated by phosphodiesterase type 4 in cultured adult rat aortic smooth muscle cells.

Zhai K, Hubert F, Nicolas V, Ji G, Fischmeister R, Leblais V - PLoS ONE (2012)

Effect of PDE inhibitors on the peak amplitude and t1/2off of β-AR-induced cytosolic cAMP signal in RASMCs.Dynamic parameters (peak amplitude, A and t1/2off, B) obtained in Figure 5 are expressed in % of isoproterenol-induced response (Control). Data are mean±SEM of 8–13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 versus Control; $ P<0.05, $$ P<0.01 and $$$ P<0.001 versus IBMX; # P<0.05, ### P<0.001 as indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475707&req=5

pone-0047826-g006: Effect of PDE inhibitors on the peak amplitude and t1/2off of β-AR-induced cytosolic cAMP signal in RASMCs.Dynamic parameters (peak amplitude, A and t1/2off, B) obtained in Figure 5 are expressed in % of isoproterenol-induced response (Control). Data are mean±SEM of 8–13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 versus Control; $ P<0.05, $$ P<0.01 and $$$ P<0.001 versus IBMX; # P<0.05, ### P<0.001 as indicated.
Mentions: To evaluate the functional contribution of the different PDE families in regulating the cAMP response to β-AR stimulation, RASMCs were incubated in the presence of PDE inhibitors during 3 min before Iso pulse. Application of PDE inhibitors alone, with the exception of BAY and BRL, induced a slight but significant increase in basal FRET ratio, which was maximal with 50 µM MIMX (the PDE1 inhibitor) and 100 µM IBMX (the non-selective PDE inhibitor) (Table2). 50 µM MIMX (Figure5A), 100 nM BAY (the PDE2 inhibitor, Figure5B) and 1 µM Cil (the PDE3 inhibitor, Figure5C) had no effect on the dynamics of the Iso-induced FRET signal. 50 µM BRL (the PDE7 inhibitor, Figure5E) had only minor effects on its kinetics (with a slight increase in the time to peak of 17%, n = 9, P<0.05), whereas 10 µM Ro (the PDE4 inhibitor, Figure5D) markedly increased its amplitude (by 52%, n = 13, P<0.01) and significantly delayed its onset and recovery phases. IBMX (Figure5I) also increased the amplitude of Iso response (by 76%, n = 9, P<0.05) and dramatically prolonged its duration. The IBMX effect on the decay phase was significantly higher than that induced by Ro (t1/2off increased by 99% and 770% with Ro and IBMX, respectively, P<0.001; Figure6B). This indicates that, in RASMCs, PDE4 is an essential regulator of β-AR-elicited cytosolic cAMP signals, but that other PDEs might also contribute to this regulation.

Bottom Line: Both β(1)- and β(2)-AR antagonists decreased the signal amplitude without affecting its kinetics.PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment.This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

View Article: PubMed Central - PubMed

Affiliation: Inserm UMR-S 769, LabEx LERMIT, Châtenay-Malabry, France.

ABSTRACT

Background: We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs).

Methodology/principal findings: The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3  =  PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β(1)- and β(2)-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β(1)- and β(2)-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation.

Conclusion/significance: Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

Show MeSH
Related in: MedlinePlus