Dataset Information

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

ABSTRACT:

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 Förster 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 ?-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.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6514293 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Zhang Liang L Bouadjel Kaouter K Manoury Boris B Vandecasteele Grégoire G Fischmeister Rodolphe R Leblais Véronique V

British journal of pharmacology 20190424 11

<h4>Background and purpose</h4>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]<sub>i</sub> , [cGMP]<sub>i</sub> ) in cultured rat aortic smooth muscle cells (RASMCs).<h4>Experimental appro ...[more]

PMID: 30825186

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Project description:BACKGROUND:The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-?B ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-?B signalling is essential for pro-calcific activation of the underlying SMCs. METHODS:For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25?ng/ml?±?5?ng/ml TRAIL) for 72?h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5?ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RESULTS:Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-?B/p52 and canonical NF-?B/p65 pathways in both cell types. In RANKL?±?TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-?B/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-?B2 gene (the molecular forerunner to NF-?B/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SUMMARY:These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-?B pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.

Dataset Information

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

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

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

Similar Datasets

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