Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells

The influence of cell confluence on the β-adrenoceptor (β-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. β-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the β-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100μM) induced a similar saturating response in both LD and HD cells. A β1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a β2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a β-AR ligand, identified two binding sites in HD cells, corresponding to β1- and β2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to β2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10μM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1μM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the β-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both β1- and β2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only β2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the β1-AR, a lower cAMP-PDE activity and a h