Cyclic nucleotide signalling compartmentation by phosphodiesterases in cultured vascular smooth muscle cells.

BACKGROUND AND PURPOSE: PDE upregulation is associated with several vascular diseases and better understanding of the respective role of each PDE family in controlling subcellular pools of cyclic nucleotides in vascular cells is needed. We investigated the respective role of PDE1, PDE5 and PDE9 in controlling intracellular cAMP and/or cGMP concentrations ([cAMP]i , [cGMP]i ) in cultured rat aortic smooth muscle cells (RASMCs).

EXPERIMENTAL APPROACH: By using a pharmacological approach with selective PDE1, PDE5 and PDE9 inhibitors (PF-04471141, sildenafil and PF-04447943, respectively), cAMP- and cGMP-PDE activities were measured by a radioenzymatic assay in protein extracts. Real-time [cAMP]i and [cGMP]i were recorded by Förster resonance energy transfer (FRET)-imaging in single living cells, and cell proliferation was assessed in FBS-stimulated cells.

KEY RESULTS: Biochemical assay revealed that PDE1, PDE5 and PDE9 represent the major cGMP-hydrolysing activity in RASMCs. Basal PDE1 exerted a functional role in degrading in situ the cGMP produced in response to the particulate GC activation by the C-type Natriuretic Peptide (CNP). Interestingly, upon high intracellular Ca2+ concentration, PDE1 also regulated the nitric oxide (NO)/soluble GC-dependent cGMP response, as well as the β-adrenoceptor-mediated cAMP response. PDE5 exerted a major role in degrading cGMP produced by NO and the natriuretic peptides. PDE9 only regulated the NO-induced [cGMP]i increase. All three PDEs contributed differently to regulate cell proliferation under basal condition and upon cGMP-elevating stimuli.

CONCLUSIONS AND IMPLICATIONS: Our work underscores the distinct role of PDE1, PDE5 and PDE9 in locally regulating the [cAMP]i and [cGMP]i , in vascular smooth muscle cells, strengthening the concept of PDEs as key actors of cyclic nucleotide subcellular compartmentation.