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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 steady-state activation of β-AR on cytosolic [cAMP] in RASMCs.Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in cultured RAMSCs cells incubated with cumulative increasing concentrations of isoproterenol (0.1 nM to 1 µM). A: Representative cAMP signals monitored in one cell. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph. B: Concentration-response curve of isoproterenol effect as shown in A. Data are mean±SEM of 7 independent cells.
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pone-0047826-g003: Effect of steady-state activation of β-AR on cytosolic [cAMP] in RASMCs.Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in cultured RAMSCs cells incubated with cumulative increasing concentrations of isoproterenol (0.1 nM to 1 µM). A: Representative cAMP signals monitored in one cell. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph. B: Concentration-response curve of isoproterenol effect as shown in A. Data are mean±SEM of 7 independent cells.

Mentions: We then evaluated the functional contribution of the different PDE isoforms in controlling intracellular cAMP concentration ([cAMP]i) in RASMCs. [cAMP]i changes in response to Iso were monitored in real-time by fluorescence imaging using the FRET-based cAMP sensor Epac1-camps. As illustrated on the images in Figure3A and Figure4A, the CFP/YFP ratio fluorescence was distributed throughout the cytosol in cells expressing Epac1-camps, indicating a cytosolic localization of the probe. Cumulative increasing concentrations of Iso (0.1 nM to 1 µM) induced a significant increase in CFP/YFP ratio in a concentration-dependent manner, with a maximum response of 27.9±2.0% and an EC50 value of 18.1±2.2 nM (n = 7) (Figure3B). These alterations of FRET signal reflect the production of cytosolic cAMP upon β-AR stimulation. To analyze the dynamics of this cAMP signal, Iso was then applied transiently at the concentration of 0.1 µM. As shown in Figure4, a 15 s-pulse of Iso induced a rapid increase in the CFP/YFP ratio to reach a maximum of 14.1±0.7% at 48.7±1.4 s (n = 124) before returning to baseline with a t1/2off of 51.5±3.0 s. Thus, a short β-AR stimulation induced a transient increase in cytosolic [cAMP]i, suggesting that cAMP is rapidly metabolized in RASMCs, most likely through cAMP hydrolysis by PDEs.


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 steady-state activation of β-AR on cytosolic [cAMP] in RASMCs.Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in cultured RAMSCs cells incubated with cumulative increasing concentrations of isoproterenol (0.1 nM to 1 µM). A: Representative cAMP signals monitored in one cell. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph. B: Concentration-response curve of isoproterenol effect as shown in A. Data are mean±SEM of 7 independent cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047826-g003: Effect of steady-state activation of β-AR on cytosolic [cAMP] in RASMCs.Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in cultured RAMSCs cells incubated with cumulative increasing concentrations of isoproterenol (0.1 nM to 1 µM). A: Representative cAMP signals monitored in one cell. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph. B: Concentration-response curve of isoproterenol effect as shown in A. Data are mean±SEM of 7 independent cells.
Mentions: We then evaluated the functional contribution of the different PDE isoforms in controlling intracellular cAMP concentration ([cAMP]i) in RASMCs. [cAMP]i changes in response to Iso were monitored in real-time by fluorescence imaging using the FRET-based cAMP sensor Epac1-camps. As illustrated on the images in Figure3A and Figure4A, the CFP/YFP ratio fluorescence was distributed throughout the cytosol in cells expressing Epac1-camps, indicating a cytosolic localization of the probe. Cumulative increasing concentrations of Iso (0.1 nM to 1 µM) induced a significant increase in CFP/YFP ratio in a concentration-dependent manner, with a maximum response of 27.9±2.0% and an EC50 value of 18.1±2.2 nM (n = 7) (Figure3B). These alterations of FRET signal reflect the production of cytosolic cAMP upon β-AR stimulation. To analyze the dynamics of this cAMP signal, Iso was then applied transiently at the concentration of 0.1 µM. As shown in Figure4, a 15 s-pulse of Iso induced a rapid increase in the CFP/YFP ratio to reach a maximum of 14.1±0.7% at 48.7±1.4 s (n = 124) before returning to baseline with a t1/2off of 51.5±3.0 s. Thus, a short β-AR stimulation induced a transient increase in cytosolic [cAMP]i, suggesting that cAMP is rapidly metabolized in RASMCs, most likely through cAMP hydrolysis by PDEs.

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