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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 transient activation of β-AR on submembrane [cAMP] in RASMCs.Submembrane cAMP measurements were conducted in cultured RASMCs cells using the plasma membrane-targeted FRET-based cAMP sensor pm-Epac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s). A: Submembrane localization of pm-Epac2-camps was ascertained by recording the CFP emission. B: Variation of the CFP/YFP ratio monitored in one cell. C: Dynamic parameters (peak amplitude, tmax, t1/2on, and t1/2off) of isoproterenol-induced FRET signal as shown in B. Data are mean±SEM of 23 independent cells.
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pone-0047826-g008: Effect of transient activation of β-AR on submembrane [cAMP] in RASMCs.Submembrane cAMP measurements were conducted in cultured RASMCs cells using the plasma membrane-targeted FRET-based cAMP sensor pm-Epac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s). A: Submembrane localization of pm-Epac2-camps was ascertained by recording the CFP emission. B: Variation of the CFP/YFP ratio monitored in one cell. C: Dynamic parameters (peak amplitude, tmax, t1/2on, and t1/2off) of isoproterenol-induced FRET signal as shown in B. Data are mean±SEM of 23 independent cells.

Mentions: To directly monitor cAMP dynamics in the submembrane compartment, we used the modified FRET-based cAMP sensor, pm-Epac2-camps, which was effectively targeted to the plasma membrane (Figure8A). As shown in Figure8B, pm-Epac2-camps generated a FRET response upon transient stimulation with Iso at the concentration of 10 nM. The FRET signal reached a maximum of 11.9±1.3% at 99.8±4.4 s (n = 23 cells) before returning to baseline with a t1/2off of 78.4±8.8 s (Figure8C). We then investigated the role of PDE3 and PDE4 families in regulating submembrane cAMP produced by β-AR stimulation. Application of 1 µM Cil or 10 µM Ro slightly increased the basal FRET ratio (Table3). Both PDE3 and PDE4 inhibitors significantly affected the Iso-induced FRET signal by increasing its amplitude (by 51% and 38%, respectively) and delaying its recovery phase (Figure9A and B). When Cil and Ro were applied concomitantly, the basal FRET ratio was markedly enhanced (Table3) and the Iso response was significantly increased in amplitude by 59% (P<0.05, n = 8) and dramatically prolonged in duration (t1/2off increased by 548%, compared to 150% and 85% with Cil and Ro alone, respectively, P<0.001; Figure9C). It should be noted that the effect of PDE inhibitors on the amplitude of Iso response was underestimated here given their effect on the basal FRET ratio. Altogether, these results suggest that, unlike in the cytosol, both PDE3 and PDE4 control submembrane cAMP concentration in RASMCs.


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 transient activation of β-AR on submembrane [cAMP] in RASMCs.Submembrane cAMP measurements were conducted in cultured RASMCs cells using the plasma membrane-targeted FRET-based cAMP sensor pm-Epac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s). A: Submembrane localization of pm-Epac2-camps was ascertained by recording the CFP emission. B: Variation of the CFP/YFP ratio monitored in one cell. C: Dynamic parameters (peak amplitude, tmax, t1/2on, and t1/2off) of isoproterenol-induced FRET signal as shown in B. Data are mean±SEM of 23 independent cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047826-g008: Effect of transient activation of β-AR on submembrane [cAMP] in RASMCs.Submembrane cAMP measurements were conducted in cultured RASMCs cells using the plasma membrane-targeted FRET-based cAMP sensor pm-Epac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s). A: Submembrane localization of pm-Epac2-camps was ascertained by recording the CFP emission. B: Variation of the CFP/YFP ratio monitored in one cell. C: Dynamic parameters (peak amplitude, tmax, t1/2on, and t1/2off) of isoproterenol-induced FRET signal as shown in B. Data are mean±SEM of 23 independent cells.
Mentions: To directly monitor cAMP dynamics in the submembrane compartment, we used the modified FRET-based cAMP sensor, pm-Epac2-camps, which was effectively targeted to the plasma membrane (Figure8A). As shown in Figure8B, pm-Epac2-camps generated a FRET response upon transient stimulation with Iso at the concentration of 10 nM. The FRET signal reached a maximum of 11.9±1.3% at 99.8±4.4 s (n = 23 cells) before returning to baseline with a t1/2off of 78.4±8.8 s (Figure8C). We then investigated the role of PDE3 and PDE4 families in regulating submembrane cAMP produced by β-AR stimulation. Application of 1 µM Cil or 10 µM Ro slightly increased the basal FRET ratio (Table3). Both PDE3 and PDE4 inhibitors significantly affected the Iso-induced FRET signal by increasing its amplitude (by 51% and 38%, respectively) and delaying its recovery phase (Figure9A and B). When Cil and Ro were applied concomitantly, the basal FRET ratio was markedly enhanced (Table3) and the Iso response was significantly increased in amplitude by 59% (P<0.05, n = 8) and dramatically prolonged in duration (t1/2off increased by 548%, compared to 150% and 85% with Cil and Ro alone, respectively, P<0.001; Figure9C). It should be noted that the effect of PDE inhibitors on the amplitude of Iso response was underestimated here given their effect on the basal FRET ratio. Altogether, these results suggest that, unlike in the cytosol, both PDE3 and PDE4 control submembrane cAMP concentration in RASMCs.

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