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Phosphodiesterases (PDE) type 3 and 4 promote vasoconstriction by hydrolysing cAMP. In experimental heart failure (HF), PDE3 makes PDE4 redundant in aorta, but it is not known if this occurs in resistance vessels, such as mesenteric artery. As PDE2 is increased in the failing myocardium, its possible role in the vasculature also needs to be addressed. Here, the function of PDE2, PDE3 and PDE4 in rat mesenteric arteries was characterized in experimental HF. Mesenteric arteries were isolated from rats sacrificed 22 weeks after surgical stenosis of the ascending aorta (HF), or Sham surgery. PDE inhibitors were used to probe isoenzyme contributions in enzymatic and isometric tension assays. PDE2 and PDE4 activities, but not PDE3 activity, facilitate contraction produced by the thromboxane analogue U46619 in Sham arteries, while in HF all three isoenzymes contribute to this response. NO synthase inhibition by L-NAME abolished the action of the PDE2 inhibitor. L-NAME eliminated the contribution of PDE4 in HF, but unmasked a contribution for PDE3 in Sham. PDE3 and PDE4 activities attenuated relaxant response to β-adrenergic stimulation in Sham and HF. PDE2 did not participate in cAMP or cGMP-mediated relaxant responses. PDE3 and PDE4 cAMP-hydrolysing activities were smaller in HF mesenteric arteries, while PDE2 activity was scarce in both groups. Endothelial cells and arterial myocytes displayed PDE2 immunolabelling. We highlight that, by contrast with previous observations in aorta, PDE4 participates equally as PDE3 in contracting mesenteric artery in HF. PDE2 activity emerges as a promoter of contractile response that is preserved in HF., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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: 3·10 3 cells/cm 2 ) or high density (HD: 3·10 4 cells/cm 2 ) 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 β 1 -AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a β 2 -AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [ 125 I]-ICYP binding experiments with betaxolol, a β-AR ligand, identified two binding sites in HD cells, corresponding to β 1 - and β 2 -ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to β 2 -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 β 1 - and β 2 -AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only β 2 -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 β 1 -AR, a lower cAMP-PDE activity and a higher basal [cAMP] i . This study highlights the critical role of the cellular environment in controlling the vascular β-AR signalling., (Copyright © 2017. Published by Elsevier Inc.)

Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac and vascular muscle functions. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium and vascular smooth muscle, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling and smooth muscle contractile tone. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important for the degradation of cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. There is experimental evidence that these PDEs, as well as other PDE families, including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure, as well as several vascular diseases. After a brief presentation of the cyclic nucleotide pathways in cardiac and vascular cells, and the major characteristics of the PDE superfamily, this review will focus on the current use of PDE inhibitors in cardiovascular diseases, and the recent research developments that could lead to better exploitation of the therapeutic potential of these enzymes in the future., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K(+) channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH)., Methods and Results: We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats., Conclusions: In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted., (© 2016 American Heart Association, Inc.)