0059: Cyclic AMP response to β-adrenergic stimulation is strongly dependent on cell confluence in vascular smooth muscle cells

The influence of cell confluence on β-adrenergic (β-AR) cAMP signal and its control by phosphodiesterases (PDE) was investigated in vascular smooth muscle cells (SMC). Cultured rat aortic SMC were plated either at low density (LD: 3.103 cells/cm2) or high density (HD: 3.104 cells/cm2) corresponding, respectively, to non-confluent or confluent cells 2 days later. The cells were infected with an adenovirus encoding the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps and imaging experiments were performed 48 h later. A brief (15 s) application of the β-AR agonist isoproterenol (Iso) induced a typical transient FRET signal, with a rapid increase, reflecting cAMP production, followed by a return to baseline. The amplitude of response to 10 or 100 nM Iso was higher in HD than LD cells, but the kinetics were similar. A β1-AR antagonist (CGP 20712, 100 nM) reduced the Iso (100 nM) response in HD but not LD cells. However, a β2-AR antagonist (ICI 118551, 5 nM) reduced the response in both conditions. To evaluate whether the absence of β1-AR response in LD cells was due to an increased PDE activity, we used the non-selective PDE inhibitor IBMX (100 μM). In LD cells pretreated with IBMX, the Iso response was maintained even after Iso washout, showing that the recovery phase is due to cAMP hydrolysis. However, CGP 20712 still did not alter the Iso response. PDE4 inhibition with Ro-20-1724 (10 μM) strongly prolonged Iso (10 nM) response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1 μM) potentiated Iso response in LD cells but had no effect in HD cells, even at a 100 nM Iso concentration. We conclude that in HD cells, Iso response involves both β1- and β2-AR stimulation and is controlled by PDE4 but not PDE3, whereas in LD cells, Iso response involves only β2-AR stimulation and is controlled by both PDE3 and PDE4. Thus, β-AR-mediated cAMP signal in vascular SMC is strongly dependent on the confluence of the cells.