Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of β1-AR and β2-AR antagonists on β-AR-induced cytosolic cAMP signals in RASMCs.Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence or presence of β-AR antagonists and PDE4 inhibitor. A, B: Effect of β1-AR (100 nM CGP-20712A, CGP; A) and β2-AR (5 nM ICI 118,551, ICI; B) antagonists on Iso response. C, D: Effect of PDE4 inhibitor (10 µM Ro) on Iso response obtained in the presence of β1-AR (Iso+CGP; C) or β2-AR (Iso+ICI; D) antagonists. Top and lower panels represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are mean±SEM of 7-13 independent cells as indicated. * P<0.05, ** P<0.01 versus Iso (A, B), Iso+CGP (C) or Iso+ICI (D).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475707&req=5

pone-0047826-g007: Effect of β1-AR and β2-AR antagonists on β-AR-induced cytosolic cAMP signals in RASMCs.Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence or presence of β-AR antagonists and PDE4 inhibitor. A, B: Effect of β1-AR (100 nM CGP-20712A, CGP; A) and β2-AR (5 nM ICI 118,551, ICI; B) antagonists on Iso response. C, D: Effect of PDE4 inhibitor (10 µM Ro) on Iso response obtained in the presence of β1-AR (Iso+CGP; C) or β2-AR (Iso+ICI; D) antagonists. Top and lower panels represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are mean±SEM of 7-13 independent cells as indicated. * P<0.05, ** P<0.01 versus Iso (A, B), Iso+CGP (C) or Iso+ICI (D).

Mentions: To evaluate the contribution of β1- and β2-AR subtypes in cytosolic cAMP production elicited by Iso, RASMCs were incubated in the presence of β1- or β2-AR antagonists during 3 min before the Iso pulse. The selective β1-AR antagonist CGP-20712A was used at a concentration of 100 nM, which is about 100-fold higher than its binding affinity [29], [30]. At this concentration, CGP-20712A did not modify the FRET signal induced by a selective β2-AR agonist (FigureS3), confirming its selectivity towards β1-ARs in our experimental conditions. By contrast, CGP-20712A significantly decreased the amplitude of Iso response by 39% without affecting its kinetics (Figure7A), indicating that β1-ARs are involved in this response. To antagonize β2-ARs, we used ICI 118,551, which exhibits a binding affinity for β2-ARs of around 1 nM and a selectivity ratio for β2-ARs over β1-ARs of about 70 [30]. We evaluated the effect of different concentrations of ICI 118,551 (1, 5, 10 and 100 nM) on FRET signal induced by Iso in RASMCs. ICI 118,551 significantly reduced the amplitude of Iso response in a concentration-dependent manner [by 14% (n = 6), 48% (n = 7), 55% (n = 5) and 90% (n = 9) at 1, 5, 10 and 100 nM, respectively], without affecting its kinetics (FigureS4 and Figure7B for ICI 118,551 at 5 nM), indicating that β2-ARs are involved in this response. In the following experiments, we decided to use ICI 118,551 at 5 nM, a concentration which should preserve its selectivity towards β2-ARs according to its binding affinities.


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 β1-AR and β2-AR antagonists on β-AR-induced cytosolic cAMP signals in RASMCs.Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence or presence of β-AR antagonists and PDE4 inhibitor. A, B: Effect of β1-AR (100 nM CGP-20712A, CGP; A) and β2-AR (5 nM ICI 118,551, ICI; B) antagonists on Iso response. C, D: Effect of PDE4 inhibitor (10 µM Ro) on Iso response obtained in the presence of β1-AR (Iso+CGP; C) or β2-AR (Iso+ICI; D) antagonists. Top and lower panels represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are mean±SEM of 7-13 independent cells as indicated. * P<0.05, ** P<0.01 versus Iso (A, B), Iso+CGP (C) or Iso+ICI (D).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047826-g007: Effect of β1-AR and β2-AR antagonists on β-AR-induced cytosolic cAMP signals in RASMCs.Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence or presence of β-AR antagonists and PDE4 inhibitor. A, B: Effect of β1-AR (100 nM CGP-20712A, CGP; A) and β2-AR (5 nM ICI 118,551, ICI; B) antagonists on Iso response. C, D: Effect of PDE4 inhibitor (10 µM Ro) on Iso response obtained in the presence of β1-AR (Iso+CGP; C) or β2-AR (Iso+ICI; D) antagonists. Top and lower panels represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are mean±SEM of 7-13 independent cells as indicated. * P<0.05, ** P<0.01 versus Iso (A, B), Iso+CGP (C) or Iso+ICI (D).
Mentions: To evaluate the contribution of β1- and β2-AR subtypes in cytosolic cAMP production elicited by Iso, RASMCs were incubated in the presence of β1- or β2-AR antagonists during 3 min before the Iso pulse. The selective β1-AR antagonist CGP-20712A was used at a concentration of 100 nM, which is about 100-fold higher than its binding affinity [29], [30]. At this concentration, CGP-20712A did not modify the FRET signal induced by a selective β2-AR agonist (FigureS3), confirming its selectivity towards β1-ARs in our experimental conditions. By contrast, CGP-20712A significantly decreased the amplitude of Iso response by 39% without affecting its kinetics (Figure7A), indicating that β1-ARs are involved in this response. To antagonize β2-ARs, we used ICI 118,551, which exhibits a binding affinity for β2-ARs of around 1 nM and a selectivity ratio for β2-ARs over β1-ARs of about 70 [30]. We evaluated the effect of different concentrations of ICI 118,551 (1, 5, 10 and 100 nM) on FRET signal induced by Iso in RASMCs. ICI 118,551 significantly reduced the amplitude of Iso response in a concentration-dependent manner [by 14% (n = 6), 48% (n = 7), 55% (n = 5) and 90% (n = 9) at 1, 5, 10 and 100 nM, respectively], without affecting its kinetics (FigureS4 and Figure7B for ICI 118,551 at 5 nM), indicating that β2-ARs are involved in this response. In the following experiments, we decided to use ICI 118,551 at 5 nM, a concentration which should preserve its selectivity towards β2-ARs according to its binding affinities.

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