A pathophysiological role of PDE3 in allergic airway inflammation.

Phosphodiesterase 3 (PDE3) and PDE4 regulate levels of cyclic AMP, which are critical in various cell types involved in allergic airway inflammation. Although PDE4 inhibition attenuates allergic airway inflammation, reported side effects preclude its application as an antiasthma drug in humans. Case reports showed that enoximone, which is a smooth muscle relaxant that inhibits PDE3, is beneficial and lifesaving in status asthmaticus and is well tolerated. However, clinical observations also showed antiinflammatory effects of PDE3 inhibition. In this study, we investigated the role of PDE3 in a house dust mite-driven (HDM-driven) allergic airway inflammation (AAI) model that is characterized by T helper 2 cell activation, eosinophilia, and reduced mucosal barrier function. Compared with wild-type (WT) littermates, mice with a targeted deletion of the PDE3A or PDE3B gene showed significantly reduced HDM-driven AAI. Therapeutic intervention in WT mice showed that all hallmarks of HDM-driven AAI were abrogated by the PDE3 inhibitors enoximone and milrinone. Importantly, we found that enoximone also reduced the upregulation of the CD11b integrin on mouse and human eosinophils in vitro, which is crucial for their recruitment during allergic inflammation. This study provides evidence for a hitherto unknown antiinflammatory role of PDE3 inhibition in allergic airway inflammation and offers a potentially novel treatment approach.

Regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2.

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.

Female infertility in PDE3A(-/-) mice: polo-like kinase 1 (Plk1) may be a target of protein kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A(-/-) mice.

Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G2/M-arrested PDE3A(-/-) murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A(-/-) oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A(-/-) oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A(-/-) oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A(-/-) mice.

Phosphodiesterase 3 (PDE3): structure, localization and function.

Cyclic adenosine 3'5'-monophosphate (cAMP) and cyclic guanosine 3'5'-monophosphate (cGMP) are critical intracellular messengers involved in transduction of signals generated by a wide variety of extracellular stimuli, including growth factors, cytokines, peptide hormones, light and neurotransmitters. These messengers modulate many fundamental biological processes, including myocardial contractility, platelet aggregation, vascular smooth muscle relaxation, proliferation and apoptosis, etc. Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, and are important in regulating intracellular concentrations and biological actions of these signal-transducing molecules. These enzymes contain at least 11 highly regulated and structurally related gene families (PDE1-11). In this review, we will discuss some general information of PDEs and then focus on PDE3 gene family, including the molecular biology, structure, function and potential as therapeutic targets. Furthermore, we show the possibilities of PDE3 as therapeutic targets in malignant tumor cells and salivary gland.

Phosphodiesterase 3B is localized in caveolae and smooth ER in mouse hepatocytes and is important in the regulation of glucose and lipid metabolism.

Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor gamma, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes.

Increased phosphodiesterase 3A/4B expression after angioplasty and the effect on VASP phosphorylation.

The beneficial effects of coronary angioplasty are limited by the proliferation and migration of vascular smooth muscle cells leading to restenosis. We hypothesized that increased activity of phosphodiesterase (PDE) after angioplasty in response to growth factors such as platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF), leads to reduced cAMP levels, which, in turn, may contribute to vascular smooth muscle cell proliferation. In rats subjected to angioplasty, aortic expression and activity of PDE3/PDE4 were increased within 24 h and associated with reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP-dependent protein kinase A (PKA). Inhibition of PDE3 increased VASP phosphorylation in aortic rings from rats subjected to angioplasty, whereas inhibition of PDE4 or stimulation of adenylate cyclase with isoproterenol was without effect; however, combined inhibition of PDE3 and PDE4 produced a synergistic effect on VASP phosphorylation. In cultured vascular smooth muscle cells, exposure to PDGF-BB resulted in increased expression of PDE3, which was prevented by an inhibitor of PI3 kinase but not by inhibitors of the MAP kinase signaling pathway. In contrast, FGF increased the expression of PDE4 in vascular smooth muscle cells but did not influence expression of PDE3. This study shows that angioplasty results in increased expression/activity of PDE, possibly arising from stimulation by PDGF-BB and FGF, and decreased cAMP levels, which may promote restenosis. These results provide a rational explanation for the beneficial effects of PDE inhibitors.

Real-time monitoring of phosphodiesterase inhibition in intact cells.

Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of PDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze PDE activity in living cells. It allows to sense cAMP gradients around particular PDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling.