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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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cAMP-PDE activity in RASMCs.cAMP-PDE activity was determined in lysates of cultured RAMSCs in the absence (vehicle) or presence of selective PDE inhibitors (PDE1: 10 or 50 µM MIMX; PDE2: 100 nM BAY; PDE3: 1 µM Cil; PDE4: 10 µM Ro; PDE7: 50 µM BRL) or a non-selective PDE inhibitor (1 mM IBMX) or a combination of several inhibitors as indicated. Results are expressed in % of cAMP-PDE activity measured in the absence of inhibitors (vehicle). Data are mean±SEM of 3–6 independent experiments. *** P<0.001 versus vehicle; ## P<0.01 and ### P<0.001 versus Ro; $$$ P<0.001 as indicated.
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pone-0047826-g002: cAMP-PDE activity in RASMCs.cAMP-PDE activity was determined in lysates of cultured RAMSCs in the absence (vehicle) or presence of selective PDE inhibitors (PDE1: 10 or 50 µM MIMX; PDE2: 100 nM BAY; PDE3: 1 µM Cil; PDE4: 10 µM Ro; PDE7: 50 µM BRL) or a non-selective PDE inhibitor (1 mM IBMX) or a combination of several inhibitors as indicated. Results are expressed in % of cAMP-PDE activity measured in the absence of inhibitors (vehicle). Data are mean±SEM of 3–6 independent experiments. *** P<0.001 versus vehicle; ## P<0.01 and ### P<0.001 versus Ro; $$$ P<0.001 as indicated.

Mentions: Total cAMP-PDE activity measured in lysates from cultured RASMCs was 37.4±7.1 pmol/min/mg protein (n = 6). To determine which PDE families contribute to this total hydrolyzing activity, the assay was also conducted in the presence of different PDE inhibitors (Figure2). Consistent with the absence of PDE2 mRNA expression, BAY (100 nM), a selective PDE2 inhibitor [23], had no effect on cAMP-PDE activity. Cil (1 µM, a selective PDE3 inhibitor [24]) and Ro (10 µM, a selective PDE4 inhibitor [25]) reduced cAMP-PDE activity by 20% and 40%, respectively. As no perfect PDE1 inhibitor is commercially available, we used MIMX which blocks PDE1 activity by interfering with its catalytic site with a low micromolar affinity and exhibits a selectivity over other PDEs of 30- to 50-fold [25]-[27]. MIMX decreased cAMP-PDE activity by 22% and 37%, when used at 10 µM and 50 µM, respectively. In the simultaneous presence of MIMX (10 µM), Cil and Ro, total cAMP-PDE activity was reduced by 75%. Increasing the concentration of MIMX to 50 µM had no further effect under this condition. The PDE7 inhibitor BRL (50 µM) [28] lowered the total cAMP-PDE activity by 16%. However, BRL had no additive inhibitory effect on the cAMP-PDE activity measured in the presence of MIMX (10 µM), Cil and Ro which questions its specificity on PDE7. Finally, the broad-spectrum PDE inhibitor IBMX (1 mM) [25] inhibited the total cAMP-PDE activity by 96%. In summary, the rank order of PDE families contributing to global cAMP-PDE activity in cultured RASMCs was PDE4> PDE3 = PDE1. PDE2 activity is absent and PDE7 activity uncertain.


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)

cAMP-PDE activity in RASMCs.cAMP-PDE activity was determined in lysates of cultured RAMSCs in the absence (vehicle) or presence of selective PDE inhibitors (PDE1: 10 or 50 µM MIMX; PDE2: 100 nM BAY; PDE3: 1 µM Cil; PDE4: 10 µM Ro; PDE7: 50 µM BRL) or a non-selective PDE inhibitor (1 mM IBMX) or a combination of several inhibitors as indicated. Results are expressed in % of cAMP-PDE activity measured in the absence of inhibitors (vehicle). Data are mean±SEM of 3–6 independent experiments. *** P<0.001 versus vehicle; ## P<0.01 and ### P<0.001 versus Ro; $$$ P<0.001 as indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475707&req=5

pone-0047826-g002: cAMP-PDE activity in RASMCs.cAMP-PDE activity was determined in lysates of cultured RAMSCs in the absence (vehicle) or presence of selective PDE inhibitors (PDE1: 10 or 50 µM MIMX; PDE2: 100 nM BAY; PDE3: 1 µM Cil; PDE4: 10 µM Ro; PDE7: 50 µM BRL) or a non-selective PDE inhibitor (1 mM IBMX) or a combination of several inhibitors as indicated. Results are expressed in % of cAMP-PDE activity measured in the absence of inhibitors (vehicle). Data are mean±SEM of 3–6 independent experiments. *** P<0.001 versus vehicle; ## P<0.01 and ### P<0.001 versus Ro; $$$ P<0.001 as indicated.
Mentions: Total cAMP-PDE activity measured in lysates from cultured RASMCs was 37.4±7.1 pmol/min/mg protein (n = 6). To determine which PDE families contribute to this total hydrolyzing activity, the assay was also conducted in the presence of different PDE inhibitors (Figure2). Consistent with the absence of PDE2 mRNA expression, BAY (100 nM), a selective PDE2 inhibitor [23], had no effect on cAMP-PDE activity. Cil (1 µM, a selective PDE3 inhibitor [24]) and Ro (10 µM, a selective PDE4 inhibitor [25]) reduced cAMP-PDE activity by 20% and 40%, respectively. As no perfect PDE1 inhibitor is commercially available, we used MIMX which blocks PDE1 activity by interfering with its catalytic site with a low micromolar affinity and exhibits a selectivity over other PDEs of 30- to 50-fold [25]-[27]. MIMX decreased cAMP-PDE activity by 22% and 37%, when used at 10 µM and 50 µM, respectively. In the simultaneous presence of MIMX (10 µM), Cil and Ro, total cAMP-PDE activity was reduced by 75%. Increasing the concentration of MIMX to 50 µM had no further effect under this condition. The PDE7 inhibitor BRL (50 µM) [28] lowered the total cAMP-PDE activity by 16%. However, BRL had no additive inhibitory effect on the cAMP-PDE activity measured in the presence of MIMX (10 µM), Cil and Ro which questions its specificity on PDE7. Finally, the broad-spectrum PDE inhibitor IBMX (1 mM) [25] inhibited the total cAMP-PDE activity by 96%. In summary, the rank order of PDE families contributing to global cAMP-PDE activity in cultured RASMCs was PDE4> PDE3 = PDE1. PDE2 activity is absent and PDE7 activity uncertain.

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