Cyclic nucleotide signalling compartmentation by PDEs in cultured vascular smooth muscle cells

Background and Purpose Up-regulation of phosphodiesterases (PDEs) is associated with several vascular diseases, and better understanding of the roles of each PDE isoform 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 We used selective inhibitors of PDE1 (PF-04471141), PDE5 (sildenafil), and PDE9 (PF-04447943) to measure cAMP- and cGMP-PDE activities with a radioenzymatic assay, in RASMC extracts. Real-time [cAMP](i) and [cGMP](i) were recorded by Forster resonance energy transfer-imaging in single living cells, and cell proliferation was assessed in FBS-stimulated cells. Key Results PDE1, PDE5, and PDE9 represented the major cGMP-hydrolyzing activity in RASMCs. Basal PDE1 exerted a functional role in degrading in situ the cGMP produced in response to activation of particulate GC by C-type natriuretic peptide. In high intracellular Ca2+ concentrations, PDE1 also regulated the NO/soluble GC-dependent cGMP response, as well as the beta-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 conditions and upon cGMP-elevating stimuli. Conclusions and Implications Our data emphasize the distinct roles of PDE1, PDE5, and PDE9 in local regulation of [cAMP](i) and [cGMP](i), in vascular smooth muscle cells, strengthening the concept of PDEs as key actors in the subcellular compartmentation of cyclic nucleotides.