Effect of transient activation of β-AR on cytosolic [cAMP] in RASMCs.

<p>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). <b><i>A</i></b>: 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><i>B</i></b>: Dynamic parameters (peak amplitude, t_max, t_1/2on, and t_1/2off) of isoproterenol-induced FRET signal as shown in <i>A</i>. Data are mean±SEM of 124 independent cells.</p

cAMP-PDE activity in RASMCs.

<p>cAMP-PDE activity was determined in lysates of cultured RAMSCs in the absence (<i>vehicle</i>) 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 (<i>vehicle</i>). Data are mean±SEM of 3–6 independent experiments. *** P<0.001 <i>versus</i> vehicle;^## P<0.01 and ^### P<0.001 <i>versus</i> Ro; ^$$$ P<0.001 as indicated.</p

Effect of PDE inhibitors on the peak amplitude and t_1/2off of β-AR-induced cytosolic cAMP signal in RASMCs.

<p>Dynamic parameters (peak amplitude, <b><i>A</i></b> and t_1/2off, <b><i>B</i></b>) obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047826#pone-0047826-g005" target="_blank">Figure 5</a> are expressed in % of isoproterenol-induced response (<i>Control</i>). Data are mean±SEM of 8–13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 <i>versus</i> Control; ^$ P<0.05, ^$$ P<0.01 and ^$$$ P<0.001 <i>versus</i> IBMX; ^# P<0.05, ^### P<0.001 as indicated.</p

Effect of PDE inhibition on β-AR-induced submembrane cAMP signal in RASMCs.

<p>Submembrane cAMP measurements were conducted in cultured RASMCs cells using the FRET-based cAMP sensor pmEpac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s) after a pre-treatment in the absence (□) or presence (▪) of one selective PDE family inhibitor (<b><i>A</i></b>: 1 µM Cil for PDE3; <b><i>B</i></b>: 10 µM Ro for PDE4) or a combination of 10 µM Ro + 1 µM Cil (<b><i>C</i></b>). <i>Top</i> and <i>lower panels</i> represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are mean ± SEM of 8-14 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 <i>versus</i> Iso.</p

Effect of PDE inhibition on basal cytosolic cAMP signal in RASMCs.

<p>Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps. The % increase in CFP/YFP ratio induced by the different PDE inhibitors or the vehicle was calculated after a 3 min-incubation. Data are mean±SEM of 8–13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 <i>versus</i> vehicle; ^# P<0.05 <i>versus</i> Ro.</p

Expression analysis of mRNA encoding cAMP-PDE isoforms in RASMCs.

<p>RT-qPCR reactions were carried out on mRNAs isolated from RASMCs. The expression of PDE1A (1A), PDE1B (1B), PDE1C (1C), PDE2A (2A), PDE3A (3A), PDE3B (3B), PDE4A (4A), PDE4B (4B), PDE4C (4C), PDE4D (4D), PDE7A (7A), PDE7B (7B), PDE8A (8A) and PDE8B (8B) was analyzed. <b><i>A</i></b>: PCR products were resolved by electrophoresis on a 3% agarose gel. Shown is the picture of a representative gel stained with GelRed®. Position of molecular weight markers is indicated in base pairs (Bp). <b><i>B</i></b>: mRNA expression was expressed using the 2^ΔCt method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047826#s2" target="_blank">Materials and methods</a>. Data are mean±SEM of 9 experiments. n.d. not detectable.</p

Effect of PDE inhibition on basal submembrane cAMP signal in RASMCs.

<p>Submembrane cAMP measurements were conducted in cultured RASMCs cells using the FRET-based cAMP sensor pm-Epac2-camps. The % increase in CFP/YFP ratio induced by the different PDE inhibitors or the vehicle was calculated after a 3 min-incubation. Data are mean±SEM of 8–14 independent cells as indicated. *** P<0.001 <i>versus</i> vehicle; ^## P<0.01 <i>versus</i> Cil and <i>versus</i> Ro.</p

Effect of transient activation of β-AR on submembrane [cAMP] in RASMCs.

<p>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). <b><i>A</i></b>: Submembrane localization of pm-Epac2-camps was ascertained by recording the CFP emission. <b><i>B</i></b>: Variation of the CFP/YFP ratio monitored in one cell. <b><i>C</i></b>: Dynamic parameters (peak amplitude, t_max, t_1/2on, and t_1/2off) of isoproterenol-induced FRET signal as shown in <i>B</i>. Data are mean±SEM of 23 independent cells.</p

Effect of steady-state activation of β-AR on cytosolic [cAMP] in RASMCs.

<p>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). <b><i>A</i></b>: 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><i>B</i></b>: Concentration-response curve of isoproterenol effect as shown in <i>A</i>. Data are mean±SEM of 7 independent cells.</p

Effect of β₁-AR and β₂-AR antagonists on β-AR-induced cytosolic cAMP signals in RASMCs.

<p>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. <b><i>A, B</i></b>: Effect of β₁-AR (100 nM CGP-20712A, <i>CGP</i>; <b><i>A</i></b>) and β₂-AR (5 nM ICI 118,551, <i>ICI</i>; <b><i>B</i></b>) antagonists on Iso response. <b><i>C, D</i></b>: Effect of PDE4 inhibitor (10 µM Ro) on Iso response obtained in the presence of β₁-AR (<i>Iso+CGP</i>; <b><i>C</i></b>) or β₂-AR (<i>Iso+ICI</i>; <b><i>D</i></b>) antagonists. <i>Top</i> and <i>lower panels</i> 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 <i>versus</i> Iso (<i>A, B</i>), Iso+CGP (<i>C</i>) or Iso+ICI (<i>D</i>).</p