A recent status on asthmaticus multiple case report by Beute demonstrated the beneficial effects of phosphodiesterase III (PDE3) and phosphodiesterase IV (PDE4) inhibition. This chapter reviews the possible underlying mechanisms, beside the known effect, for the beneficial effects of a mixed PDE3/4 inhibitor in allergic airway inflammation. Structural cells of the lung and immune system express PDE3 and 4. PDE3 and 4 inhibition have a number of consequences related to physical function and cytokine production. The most direct effect of PDE3 inhibition being relaxation of smooth muscle cells results in bronchodilation. However, PDE3 inhibition appears to go further than a mere inhibitory activity in bronchial smooth muscle. It also affects structural cells, and more importantly, it creates an improved barrier function in endothelial cells. PDE3 and 4 inhibition therefore strengthens the immune barrier; but in addition, it modifies the cells of the immune system itself, as these also express PDE3 and 4 activity, thus changing their function. All aspects of asthma-related pathophysiology seem to be affected by PDE3 and 4 inhibition. Clinical use of a mixed PDE3/4 inhibitor in respiratory diseases is currently limited to a few studies, including life-threatening asthma in which mixed PDE3/4 inhibition has a beneficial effect.
- allergic airway inflammation
Asthma is an obstructive airway disease characterized by inflamed airways, structural and physiological abnormalities in the airways, and shortness of breath . Important primary airway cells are alveolar cells, endothelial cells, and smooth muscle cells; and secondary cells are involved in regulation of innate and adoptive immunology. Conventional treatment with inhaled corticosteroids combined with beta-adrenergic agonists supports and induces smooth muscle relaxation to reopen the inflamed airways, relieves symptoms, supports inspirational and expirational flow, and reduces inflammation . These treatment regimens were also used in the extreme severe cases of asthma like status asthmaticus and patients with bronchospasm, in some cases with only minimal effect. The treatment goal in these severe cases of acute asthma is the prompt relief of respiratory distress. The great benefit of a mixed PDE3/4 inhibitor, in these severe cases, is the induction of acute as well as long-lasting bronchodilator effects .
At the moment, there is no effective treatment for these severe asthmatic patients  and there are no clear, effective guidelines. Moreover, treatment of these patients is multidisciplinary, involving first aid physicians, intensive care physicians, anesthetists, and pulmonary physicians, requiring golden standards and treatment regimens per hospital for optimal results. There are still too many asthma deaths; numbers in US are up to nine cases per day and in the UK are up to over three cases per day. Presently, in the Netherlands, there are annually more than 60.
This review discusses the PDE3 gene family and PDE3 inhibition, traditionally used in acute, refractory heart failure. We discuss the benefits of combined PDE3 and 4 inhibition in status asthmaticus , and the possible mechanisms which may be responsible for these beneficial effects of PDE3 and 4 inhibition.
2. The PDE superfamily: important regulators of cyclic nucleotide signaling pathways and networks
Intracellular signaling via complicated regulatory networks plays a critical role during physiological cellular responses. cAMP and cGMP were the first molecules described as intracellular second messengers . They regulate multiple intracellular targets, including protein kinase A and protein kinase G, guanine nucleotide exchange proteins activated by cAMP (Epacs), cyclic nucleotide-gated ion channels, and PDE activities . Intracellular concentrations of cAMP and cGMP are regulated through their synthesis by adenylyl cyclases (ACs) and guanylyl cyclases and their degradation via cyclic nucleotide PDEs. Ten different ACs have been identified and classified into two groups . The first group consists of transmembrane enzymes which are activated by different hormones, neurotransmitters, chemokines, and cytokines in the G-protein-coupled receptor cascade . Another group of cytosolic ACs is regulated by bicarbonate and calcium ions . Whereas, cytosolic ACs are all encoded by one gene, transmembrane ACs represent a group encoded by nine different genes .
The large PDE superfamily is comprised of 11 PDE gene families (PDE1–PDE11). They specifically hydrolyze cyclic nucleotides, and can be classified according to their primary structures, tissue expression, biochemical properties, regulation, and their sensitivity to different pharmacological agents . By catalyzing the hydrolysis of cAMP and cGMP, PDEs regulate the intracellular concentrations of these critical second messengers, and consequently, their downstream signaling pathways and networks. PDEs also function as important regulators in the compartmentation of cyclic nucleotide signaling pathways and networks. Individual PDEs are targeted/recruited to specific intracellular locations, where they are incorporated into specific multiprotein regulatory complexes (“signalosomes”) through protein-protein interactions. By virtue of their localization to specific compartments, PDEs can thus regulate specific cyclic nucleotide signaling pathways .
3. PDE3 and PDE4
PDE3 is expressed in pulmonary structural cells and cells of the immune system. Lung structural cells, including smooth muscle cells, epithelial cells, and endothelial cells, express PDE3. PDE3A and B are encoded by two highly related and similarly organized genes on human chromosomes, 12p12 and 11p15 [12, 13, 14]. Both PDE3A and PDE3B hydrolyze cAMP and cGMP, with 4–10 times higher affinity (Vmax) for cAMP . Biochemical and histochemical studies of the localization of PDE3 suggested that PDE3 was associated with the sarcoplasmic reticulum, Golgi endosome, and nuclear envelope in cardiac tissue . PDE3 plays a major role in cardiac contraction by modulating cAMP-dependent phosphorylation of voltage-gated Ca2+ channels and Ca2+ entry . In addition, recent studies with PDE3A and PDE3B KO mice indicate that PDE3A, not PDE3B, regulates basal contractility in mouse heart .
Kass et al. described one mechanism, whereby PDE3 might be functionally modulated by cGMP occupying the PDE3 catalytic site . PDE3 binds both cAMP and cGMP at its catalytic site with high affinity, and endogenous cGMP, generated by NO-induced activation of guanyl cyclase, can function as a competitive inhibitor of hydrolysis of cAMP by PDE3 . NO-induced cGMP/cAMP cross-talk, mediated via cGMP inhibition of cAMP hydrolysis by PDE3 which leads to increased levels of cAMP, is thought to mediate some of the effects of NO in inflammatory and lung structural cells. NO modulates pulmonary vascular tone, causing non-adrenergic-, non-cholinergic-mediated bronchodilation . Overexpression of nitric oxide synthase in both endothelial and airway epithelial cells resulted in diminished airway inflammation . Under normal conditions of NO/cGMP signaling, PDE4, with a high Km for cAMP, is thought to degrade cAMP because PDE3 with a lower Km for cAMP is inhibited by endogenous cGMP and thus can increase cAMP . PDE3-induced vasorelaxation is potentiated when NO/cGMP is suppressed as PDE3 inhibition increases both cAMP and cGMP, in which cGMP inhibits cAMP degradation. PDE4 inhibition only increases cAMP and thus is unaffected by NO/cGMP suppression . PDE3 seems to be more responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations . This suggests a beneficial effect of NO in allergic airway inflammation and urges caution in the use of NOS inhibitors . Since the first PDE3 inhibition papers in the 1990s, 11 PDE families have been identified, and presently at least four isoforms of PDE4 are known . Also, the idea of signalosomes has been postulated and partly verified .
4. Modulation of structural cells and immune cells by PDE3 and 4 inhibition
Several structural cells express PDE3. Inhibition of PDEs has a number of consequences in the pathophysiology of asthma.
4.1. Smooth muscle cells and cardiomyocytes
Cardiac muscle tissue and smooth muscles are not under conscious control. The role of PDE3 in cardiac muscle and in vascular and bronchial smooth muscle slightly differs due to regulation by different modulators and inhibitors . Vascular SMC and airway SMC are widely comparable . Reducing cAMP by PDE3 modulates contraction; PDE3 inhibition (PDE3i) leads to relaxation of vascular and airway SMC which results in vasodilation and bronchodilation due to the elevated levels of cAMP. NO activates soluble- and membrane-bound guanylate cyclases, which synthesize cyclic guanylate monophosphate (cGMP), which subsequently can serve as a competitive inhibitor of PDE3 as well as activator of cGMP protein kinases . The downstream effects of NO are limited, in part, by phosphodiesterase (PDE)-induced degradation of cGMP .
The primary mechanism behind the PDE3 regulation of myocardial physiology relates to its control of cAMP levels; inhibition of myocardial PDE3, especially PDE3A, leads to decreased cAMP breakdown, resulting in increased cAMP which mediates positive inotropic effects and increases in myocardial contractility . Although PDE3 inhibitors increase myocardial contractility and vasodilation in heart failure patients , prolonged use of the PDE3 inhibitor milrinone in these patients increased mortality was observed, most likely due to arrhythmias and cardiac arrest . Presently, milrinone has an approval for short term treatment of untreatable exacerbations of heart failure and as a chemical “bridge to transplant” . The work of Chen Yan and her colleagues suggests that the untoward effects of chronic administration of relatively high dosis of milrinone may possibly be related to long term effects of cAMP on pathological remodeling and progression of heart failure , via upregulation of inducible cAMP early repressor (ICER) and subsequent increases in cardiomyocyte apoptosis . According to this hypothesis, PDE3 inhibitors increase cAMP, leading to increased expression of ICER, which blocks transcription of PDE3. This cascade of events induced a pathological “feedback loop,” with downregulation or inhibition of PDE3 leading to increased cAMP/PKA signaling, upregulation of ICER, continued downregulation of PDE3, and enhanced apoptosis in cardiaomyocytes .
In smooth muscle cells, increased cGMP levels induce vasorelaxation. Due to effects of PDEs on hydrolysis of cGMP, PDE inhibitors play a major role in the fine-tuned regulation of this function. In addition to PDEs, NO plays an important role in vasorelaxation, perhaps, in part, by its activation of cytosolic guanylate cyclases, leading to increased production of cGMP, and subsequent inhibition of PDE3. The PDE3 inhibitor, cilostazol (Pletal), is widely used to treat intermittent claudication (IC), a lower-extremity peripheral arterial disease characterized by exercise-/ischemia-induced leg pain. It is thought that cilostazol increases walking distance and alleviates IC symptoms by cAMP-mediated vasodilation and inhibition of both platelet activation and vascular wall inflammation .
Asthma can present itself with varying levels of severity, and a particular subgroup of patients, labeled as “severe asthmatics” is characterized by the persistence of symptoms despite therapy with corticosteroids [2, 35]. Examination of bronchial airways from patients with severe asthma shows a greater amount of ASM (Airway Smooth Muscle) cell mass and of subepithelial fibrosis compared to non-severe asthmatics [36, 37]. In ex-vivo studies, ASM cells from severe asthmatics demonstrated increased cell growth and proliferation  and an increase in proliferating cell nuclear antigen, a marker of proliferation . Cultured ASM cells from mild-to-moderate asthmatics also proliferated faster than ASM cells from normal subjects . Bhavsar et al. have previously demonstrated corticosteroid insensitivity in blood monocytes and alveolar macrophages from patients with severe asthma compared to those with non-severe asthma [41, 42]. Another feature of steroid insensitivity could be the ongoing ASM cell growth because the enhanced proliferation of ASM cells from patients with mild asthma is resistant to dexamethasone . Given this perspective, it is of interest that studies with VSMC from PDE3A and PDE3B KO mice indicated that the absence of PDE3A, not PDE3B, diminished VSMC proliferation and indicated a G0 G1 cell cycle arrest . PDE3 inhibition might reduce ASM proliferation in asthmatics.
4.2. Endothelial cells
Endothelial cells play an important role in the pathophysiology of asthma. Due to the expression of adhesion molecules, they enable cells to extravasate from the bloodstream into the inflamed tissue. Endothelial cells also possess a barrier function to prevent leakage of blood fluid in the tissue. Endothelial cells express PDE3 and 4, and inhibition of PDE3 and 4 of endothelial cells inhibited eosinophil and neutrophil adherence to monolayers of endothelial cells [45, 46]. PDE3 and 4 synergistically enhance the inhibition of VCAM1 expression and eosinophil adhesion to activated-human lung microvascular endothelial cells . Inhibition of PDE3 leads to increases in cAMP which improves endothelial barrier functions and supports cell-cell junctions . BW245c, a DP receptor antagonist, increases cAMP, and enhanced endothelial barrier function in a cAMP-dependent matter via the DP receptor, a G protein coupled receptor [48, 49]. Hyperpermeability of pulmonary endothelial monolayers, evoked by thrombin or
PDE inhibition, a therapeutic approach to increase cAMP levels, was beneficial in treating capillary leakage and edema in a rat model of systemic inflammation induced by LPS . Moreover, PDE3 inhibition was compared to dobutamine treatment (β-adrenoreceptor compounds); the former showed inotropic, lusitropic, and vasodilating properties which were not seen in patients treated with dobutamine [57, 58]. In bypass surgery patients, reduced inflammatory responses were observed during PDE3 inhibition compared to placebo treatment . Furthermore, reduced TNF-α-levels, a cytokine which is increased in sepsis, were observed during PDE3 inhibition by enoximone compared to dobutamine-treated septic patients .
Hydrogen peroxide (H2O2), derived from neutrophils and other cells, supposedly is important in the development of vascular injury and thus of pulmonary edema. In a porcine pulmonary artery endothelial cell monolayer model, H2O2 increased hydraulic conductivity while selectivity was decreased. It is known that certain inhibitors of PDE isoenzymes 2, 3, and 4 could block H2O2-induced endothelial permeability . The data suggest that adenylate cyclase activation/PDE inhibition is a powerful approach to block H2O2-induced increase in endothelial permeability. This concept appears especially valuable when endothelial PDE isoenzyme patterns and PDE inhibitor profiles are matched optimally .
4.3. Epithelial cells, pneumocyte type I and type II
Human epithelial cells express PDE3 . NO and cAMP both modulate membrane water permeability via aquaporin5 expression in pneumocyte type I [63, 64]. Experimental lung edema can be attenuated by selective PDE3 and PDE4 inhibitors [50, 65, 66, 67]. In experimental pulmonary edema, PDE3 inhibition reduces the numbers of inflammatory cells in BAL . In alveolar epithelial cells, LPS-induced biosynthesis of proinflammatory cytokines is regulated by cAMP and tightly controlled by PDEs, and can be reduced by PDE inhibitors .
Inhibition of PDE3 and elevation of cAMP improve epithelial and endothelial barrier function and reduce SMC proliferation, which are interesting therapeutic targets in the future for asthma.
5. Immune cells
Mechanisms for regulation of PDE3 activity in immune cells, including dendritic cells, monocytes, B-cells, NK cells γδT cells, αβT-cells, T-cells, macrophages, eosinophils, and neutrophils, all of which express PDE3 isoforms are largely unknown (immgen database http://www.immgen.org/databrowser/index.html). Theophylline is a nonspecific PDE inhibitor . In asthmatic patients, the inflamed airway mucosa, characterized by the presence of eosinophils, IgE positive mast cells, T-cells and dendritic cells, exhibits dysregulated barrier immunity . These various inflammatory cells each have their own position in the asthma cascade. PDE3 and PDE4 are the major isoenzymes regulating IgE-stimulated mediator release from rat peritoneal mast cells . Alveolar macrophage activation can be inhibited by PDE3/PDE4 inhibitors . DC cultures were treated with a PDE4 inhibitor and with combined inhibition of PDE3 and 4; the latter resulted in a two times stronger reduction in LPS-induced TNFα release in DC cultures .
Inhibition of PDE3 and PDE4 prevents immunogen-stimulated IL-2 release from CD4 and CD8 human T-cells. Human T-cells and B-cell express PDE3 [73, 76, 77, 78]. Knock down strategies or inhibitors of PDE4B or D inhibit IL-4, IL-5, and IFNγ expression or production [79, 80, 81]. Peripheral blood mononuclear cells from atopic dermatitis patients and healthy controls show inhibition of PMA-induced proliferation due to the treatment with PDE4 inhibitors. cAMP was found to inhibit T-cell proliferation and differentiation which was linked to IL-2 [82, 83]. IL-2 activation of CD25+ T cells (Treg cells) led to a drastic upregulation in AC activity and to cAMP accumulation; an opposite significant decrease in AC activity was seen in CD25− T cells . The PDE activity remained unchanged in both cell subpopulations, suggesting that the mechanism of cAMP accumulation in stimulated Treg involves AC7 activation . Cyclic AMP is a pleiotropic regulator of cell growth and function. In T-cells, cAMP suppresses TCR-triggered proliferation and cytokine production. cAMP is also a selective modulator of the actions of the proinflammatory transcription factor NF-κB. NF-κB plays a crucial role in switching on the gene expression of inflammatory and immune mediators and is therefore an important target for therapy . cAMP is an important negative regulator of T cell activation, and increased levels of cAMP are associated with T cell hyporesponsiveness
Treatment with S-Petasin, an inhibitor of PDE3 and 4, reduced eosinophilic airway inflammation in an OVA model for asthma . Although eosinophils do not express PDE3, reduced inflammation might be an indirect consequence of elevated levels of cAMP in endothelial cells that enhance endothelial barrier function and lowered the expression of adhesion molecules [45, 47]. PDE3 inhibitors sustained increased levels of cAMP in mast cells which are inhibitory to both basophils and human lung mast cells function . Rat peritoneal mast cells showed reduced IgE-stimulated mediator release when treated with PDE3 inhibitors . The conductive players in asthma, including T-cells and DC, and the central effector cells in asthma, including eosinophils, mast cells, basophils and neutrophils, can be targeted directly or indirectly with PDE3 inhibitors.
Recently, more and more interest is seen for the “old” theophylline which is a broad PDE inhibitor . Theophylline  is a drug which targets PDE4 and, at high doses, also PDE3. However, it is a relatively weak bronchodilator at therapeutic concentrations. In patients, it is beneficial; and addition of theophylline can improve asthma control to a greater extent than beta2-agonist in patients with severe asthma . Furthermore, in asthma patients poorly controlled by steroids, low dose theophylline added to inhaled corticosteroids improves asthma control . The proposed mechanisms of action of theophylline include nonselective inhibition of PDE, antagonism of Adenosine receptors, inhibition of nuclear translocation of NF-κB, improved histone diacetylase 2, improved IL-10 secretion, induction of apoptosis of inflammatory cells (neutrophils and eosinophils) [90, 91], and inhibition of T-cell proliferation . These features are of significant importance for severe asthma with poor steroid control, in which neutrophils are found and these patients were difficult to treat . Theophylline exerted proapoptotic effects on monocyte-derived dendritic cells (DCs) and impaired DCs differentiation [90, 91].
6. PDE3 and 4 inhibition in the context of asthma
There is little literature available regarding enoximone in the context of airway disease. Bethke et al. showed that enoximone has inhibitory capacity on PDE3 and PDE4 . Fujimura et al. researched cilostazol as a PDE3 inhibitor in asthma, showing its beneficial effect on bronchial hyper-responsiveness in elder asthmatics . PDE4 inhibitors have been described in pre-clinical and clinical settings in the context of lung diseases like asthma and COPD [94, 95]. The PDE4 inhibitor roflumilast inhibits TGFβ-induced connective tissue growth factor (CTGF), collagen I and fibronectin in airway smooth muscle (ASM) cells of bronchial tissue rings . Roflumilast is approved as part of the treatment regimen for Chronic Obstructive Pulmonary Disease (COPD) . PDE3 inhibitors, including cilastozol, milrinone, and mixed PDE3/4 inhibitor enoximone, have mainly been used in the context of heart failure. Literature provides several cases with adverse effect and fatal outcome in the use of high dose PDE inhibitors for the chronic treatment of severe heart failure. A reason for this unfavorable outcome might have been that enoximone in heart failure was given in exceedingly high doses up to 2400 mg daily (31 mg/kg/dd) [98, 99, 100]; doses which were found to be extremely likely to cause severe side effects and a high mortality rate: after 6 months of treatment, at least half of the patients had died. Thus, the early research in pulmonary use has been abandoned and, since the late 90s, the sparse research into use of PDE3-inhibitors for pulmonary purposes has not led to the use of any of these drugs in the treatment of asthma. The first paper addressing actual clinical cases, in which enoximone treatment was given successfully in status asthmaticus and near fatal asthma was Beute , inspired by the resemblance between vascular and bronchial smooth muscle cell relaxation. In this paper, the doses used were considerably lower (1.15 mg/kg single dose) and the duration of administration was substantially shorter than in heart failure. Here, enoximone proved to be beneficial without side effects.
Enoximone is known as a PDE3 inhibitor that increases levels of cAMP as well as cGMP; however, in those cells where both compounds are present, cGMP will act as a competitive inhibitor on the breakdown of cAMP, thereby sustaining elevated levels of cAMP. cGMP can also be generated by nitric oxide (NO)-induced stimulation of guanylylcyclase (both abundantly present in smooth muscle cells), again impairing the breakdown of cAMP. The IC50 values of enoximone for PDE3 and PDE4 are 5.9 and 21.1 μM. The affinity of PDE3 for cAMP is 20 times higher than that of PDE4 .
These mechanisms probably allow for the favorable outcome of the relatively small doses of enoximone in Beute  and suggest an effect that exceeds its half-life.
Smooth muscle relaxation is more pronounced after administration of selective PDE3 inhibitors compared with PDE4 inhibitors. PDE3 inhibition leads to the enhancement of relaxation evoked by β2-receptor stimulation. Furthermore, simultaneous administration of siguazodan (PDE3 inhibitor) and rolipram (PDE4 inhibitor) enhances this relaxation, .
In Figure 1, both PDE3 and 4 are important in tailoring cyclic adenosine monophosphate signaling. PDE3/4 inhibitor increases intracellular cyclic adenosine monophosphate levels and has anti-inflammatory effects. Activation of a G-protein-coupled receptor (GPCR) activates adenylyl cyclase (AC) resulting in the induction of cAMP with the consequence of phosphokinase A (PKA) activation. Effect of PDE3/4 inhibition causes bronchodilation and improves endothelial and epithelial barrier function.
PDE4 is also present alongside the PDE3 isoenzyme in airway smooth muscle; the PDE3 isoenzyme is considered to predominate in airway smooth muscle, and inhibition of this enzyme leads to airway smooth muscle relaxation . Moreover, PDE3 isoenzyme A is located in the cell membrane  and presumably easy to target, and could be involved in the rapid effects of therapy (minutes or earlier) seen during the intravenous emergency treatment in the studies of Beute .
Bringing to mind once again that all the cells and mechanisms mentioned in this chapter are regulated/influenced by either PDE3, PDE4, or both, and that all these cells and mechanisms are involved in the development, maintenance, or aggravation of asthma, there is a strong case for the assumption that enoximone may have a large impact on the acute treatment of severe asthma, on various separate levels. Additional safety studies will also be required.
As discussed above, further research in PDEs appears to be advisable in order to investigate their true potential.
Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nature Reviews. Immunology. 2008; 8:183-192
Chung KF, Caramori G, Adcock IM. Inhaled corticosteroids as combination therapy with beta-adrenergic agonists in airways disease: Present and future. European Journal of Clinical Pharmacology. 2009; 65:853-871
Beute J. Emergency treatment of status asthmaticus with enoximone. British Journal of Anaesthesia. 2014. pp 1105-1108
Wenzel SE. Asthma phenotypes: The evolution from clinical to molecular approaches. Nature Medicine. 2012; 18:716-725
Sutherland EW, Rall TW. Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. The Journal of Biological Chemistry. 1958; 232:1077-1091
Francis SH, Blount MA, Corbin JD. mammalian cyclic nucleotide phosphodiesterases: Molecular mechanisms and physiological functions. Physiological Reviews. 2011; 91:651-690
Sunahara RK, Taussig R. Isoforms of mammalian adenylyl cyclase: Multiplicities of signaling. Molecular Interventions. 2002; 2:168-184
Cooper DM, Crossthwaite AJ. Higher-order organization and regulation of adenylyl cyclases. Trends in Pharmacological Sciences. 2006; 27:426-431
Kamenetsky M, Middelhaufe S, Bank EM, Levin LR, Buck J, Steegborn C. Molecular details of cAMP generation in mammalian cells: A tale of two systems. Journal of Molecular Biology. 2006; 362:623-639
Beavo JA. Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms. Physiological Reviews. 1995; 75:725-748
Maurice DH, Ke HM, Ahmad F, Wang YS, Chung J, Manganiello VC. Advances in targeting cyclic nucleotide phosphodiesterases. Nature Reviews Drug Discovery. 2014; 13:290-314
Willart MA, van Nimwegen M, Grefhorst A, Hammad H, Moons L, Hoogsteden HC, et al. Ursodeoxycholic acid suppresses eosinophilic airway inflammation by inhibiting the function of dendritic cells through the nuclear farnesoid X receptor. Allergy. 2012; 67:1501-1510
Miki T, Taira M, Hockman S, Shimada F, Lieman J, Napolitano M, et al. Characterization of the cDNA and gene encoding human PDE3B, the cGIP1 isoform of the human cyclic GMP-inhibited cyclic nucleotide phosphodiesterase family. Genomics. 1996; 36:476-485
Kasuya J, Liang SJ, Goko H, Park SH, Kato K, Xu ZD, et al. Cardiac type cGMP-inhibited phosphodiesterase (PDE3A) gene structure: Similarity and difference to adipocyte type PDE3B gene. Biochemical and Biophysical Research Communications. 2000; 268:827-834
Degerman E, Belfrage P, Manganiello VC. Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). The Journal of Biological Chemistry. 1997; 272:6823-6826
Lugnier C. Cyclic nucleotide phosphodiesterase (PDE) superfamily: A new target for the development of specific therapeutic agents. Pharmacology & Therapeutics. 2006; 109:366-398
Fischmeister R, Hartzell HC. Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells. Molecular Pharmacology. 1990; 38:426-433
Beca S, Ahmad F, Shen WX, Liu J, Makary S, Polidovitch N, et al. Phosphodiesterase Type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase Type 2a signaling complexes in mouse heart. Circulation Research. 2013; 112:289
Kass DA, Takimoto E, Nagayama T, Champion HC. Phosphodiesterase regulation of nitric oxide signaling. Cardiovascular Research. 2007; 75:303-314
Leroy MJ, Degerman E, Taira M, Murata T, Wang LH, Movsesian MA, et al. Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells. Biochemistry. 1996; 35:10194-10202
Bratt JM, Williams K, Rabowsky MF, Last MS, Franzi LM, Last JA, et al. Nitric oxide synthase enzymes in the airways of mice exposed to ovalbumin: NOS2 expression is NOS3 dependent. Mediators of Inflammation. 2010; 2010. ID: 321061
Kobayashi K, Nishimura Y, Yamashita T, Nishiuma T, Satouchi M, Yokoyama M. The effect of overexpression of endothelial nitric oxide synthase on eosinophilic lung inflammation in a murine model. International Immunopharmacology. 2006; 6:1040-1052
Yamazaki T, Anraku T, Matsuzawa S. Ibudilast, a mixed PDE3/4 inhibitor, causes a selective and nitric oxide/cGMP-independent relaxation of the intracranial vertebrobasilar artery. European Journal of Pharmacology. 2011; 650:605-611
Matthiesen K, Nielsen J. Cyclic AMP control measured in two compartments in HEK293 cells: phosphodiesterase K(M) is more important than phosphodiesterase localization. PLoS One. 2011; 6:e24392
Azevedo MF, Faucz FR, Bimpaki E, Horvath A, Levy I, de Alexandre RB, et al. Clinical and molecular genetics of the phosphodiesterases (PDEs). Endocrine Reviews. 2014; 35:195-233
Ahmad F, Degerman E, Manganiello VC. Cyclic nucleotide phosphodiesterase 3 signaling complexes. Hormone and Metabolic Research. 2012; 44:930
Berridge MJ. Introduction. Cell Signalling Biology. Module 2 Portland Press Limited. 2012:1-63
Ghofrani HA, Pepke-Zaba J, Barbera JA, Channick R, Keogh AM, Gomez-Sanchez MA, et al. Nitric oxide pathway and phosphodiesterase inhibitors in pulmonary arterial hypertension. Journal of the American College of Cardiology. 2004; 43:68S-72S
Movsesian M, Wever-Pinzon O, Vandeput F. PDE3 inhibition in dilated cardiomyopathy. Current Opinion in Pharmacology. 2011; 11:707-713
Packer M, Carver JR, Rodeheffer RJ, Ivanhoe RJ, Dibianco R, Zeldis SM, et al. Effect of oral milrinone on mortality in severe chronic heart-failure. New England Journal of Medicine. 1991; 325:1468-1475
Movsesian M, Kukreja R. Phosphodiesterase inhibition in heart failure. In: Francis SH, Conti M, Houslay MD, editors. Phosphodiesterases as Drug Targets. Berlin, Heidelberg: Springer; 2011. pp. 237-249
Yan C, Miller CL, Abe J. Regulation of phosphodiesterase 3 and inducible cAMP early repressor in the heart. Circulation Research. 2007; 100:489-501
Ding B, Abe J, Wei H, Xu HD, Che WY, Aizawa T, et al. A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis. Proceedings of the National Academy of Sciences of the United States of America. 2005; 102:14771-14776
Yasmin S, Yongge L, Junichi K. Bench to bedside. In: Cyclic Nucleotide Phosphodiesterases in Health and Disease. Boca Raton, London, New York: Taylor & Francis Group, CRC Press; 2006
Chung KF, Godard P, Adelroth E, Ayres J, Barnes N, Barnes P, et al. Difficult/therapy-resistant asthma: the need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. ERS Task Force on Difficult/Therapy-Resistant Asthma. European Respiratory Society. The European Respiratory Journal. 1999; 13:1198-1208
Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. American Journal of Respiratory and Critical Care Medicine. 2003; 167:1360-1368
Macedo P, Hew M, Torrego A, Jouneau S, Oates T, Durham A, et al. Inflammatory biomarkers in airways of patients with severe asthma compared with non-severe asthma. Clinical and Experimental Allergy. 2009; 39:1668-1676
Trian T, Benard G, Begueret H, Rossignol R, Girodet PO, Ghosh D, et al. Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma. The Journal of Experimental Medicine. 2007; 204:3173-3181
Hassan M, Jo T, Risse PA, Tolloczko B, Lemiere C, Olivenstein R, et al. Airway smooth muscle remodeling is a dynamic process in severe long-standing asthma. The Journal of Allergy and Clinical Immunology. 2010; 125:1037-1045. e3
Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, et al. Airway smooth muscle cell proliferation is increased in asthma. American Journal of Respiratory and Critical Care Medicine. 2001; 164:474-477
Bhavsar P, Hew M, Khorasani N, Torrego A, Barnes PJ, Adcock I, et al. Relative corticosteroid insensitivity of alveolar macrophages in severe asthma compared with non-severe asthma. Thorax. 2008; 63:784-790
Hew M, Bhavsar P, Torrego A, Meah S, Khorasani N, Barnes PJ, et al. Relative corticosteroid insensitivity of peripheral blood mononuclear cells in severe asthma. American Journal of Respiratory and Critical Care Medicine. 2006; 174:134-141
Roth M, Johnson PR, Borger P, Bihl MP, Rudiger JJ, King GG, et al. Dysfunctional interaction of C/EBPalpha and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells. The New England Journal of Medicine. 2004; 351:560-574
Begum N, Hockman S, Manganiello VC. Phosphodiesterase 3A (PDE3A) deletion suppresses proliferation of cultured murine vascular smooth muscle cells (VSMCs) via inhibition of mitogen-activated protein kinase (MAPK) signaling and alterations in critical cell cycle regulatory proteins. The Journal of Biological Chemistry. 2011; 286:26238-26249
Blease K, Burke-Gaffney A, Hellewell PG. Modulation of cell adhesion molecule expression and function on human lung microvascular endothelial cells by inhibition of phosphodiesterases 3 and 4. British Journal of Pharmacology. 1998; 124:229-237
Wright LC, Seybold J, Robichaud A, Adcock IM, Barnes PJ. Phosphodiesterase expression in human epithelial cells. The American Journal of Physiology. 1998; 275:L694-L700
Noda K, Zhang J, Fukuhara S, Kunimoto S, Yoshimura M, Mochizuki N. Vascular endothelial-cadherin stabilizes at cell-cell junctions by anchoring to circumferential actin bundles through alpha- and beta-catenins in cyclic AMP-Epac-Rap1 signal-activated endothelial cells. Molecular Biology of the Cell. 2010; 21:584-596
Kobayashi K, Tsubosaka Y, Hori M, Narumiya S, Ozaki H, Murata T. Prostaglandin D2-DP signaling promotes endothelial barrier function via the cAMP/PKA/Tiam1/Rac1 pathway. Arteriosclerosis, Thrombosis, and Vascular Biology. 2013; 33:565-571
Polson JB, Strada SJ. Cyclic nucleotide phosphodiesterases and vascular smooth muscle. Annual Review of Pharmacology and Toxicology. 1996; 36:403-427
Suttorp N, Ehreiser P, Hippenstiel S, Fuhrmann M, Krull M, Tenor H, et al. Hyperpermeability of pulmonary endothelial monolayer: protective role of phosphodiesterase isoenzymes 3 and 4. Lung. 1996; 174:181-194
Singleton PA, Dudek SM, Chiang ET, Garcia JG. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin. The FASEB Journal. 2005; 19:1646-1656
Hammad H, de Heer HJ, Soullie T, Hoogsteden HC, Trottein F, Lambrecht BN. Prostaglandin D2 inhibits airway dendritic cell migration and function in steady state conditions by selective activation of the D prostanoid receptor 1. Journal of Immunology. 2003; 171:3936-3940
Idzko M, Hammad H, van Nimwegen M, Kool M, Muller T, Soullie T, et al. Local application of FTY720 to the lung abrogates experimental asthma by altering dendritic cell function. The Journal of Clinical Investigation. 2006; 116:2935-2944
Idzko M, Hammad H, van Nimwegen M, Kool M, Vos N, Hoogsteden HC, et al. Inhaled iloprost suppresses the cardinal features of asthma via inhibition of airway dendritic cell function. The Journal of Clinical Investigation. 2007; 117:464-472
Konya V, Sturm EM, Schratl P, Beubler E, Marsche G, Schuligoi R, et al. Endothelium-derived prostaglandin I(2) controls the migration of eosinophils. The Journal of Allergy and Clinical Immunology. 2010; 125:1105-1113
Schick MA, Wunder C, Wollborn J, Roewer N, Waschke J, Germer CT, et al. Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation. The Journal of Physiology. 2012; 590:2693-2708
Lehtonen LA, Antila S, Pentikainen PJ. Pharmacokinetics and pharmacodynamics of intravenous inotropic agents. Clinical Pharmacokinetics. 2004; 43:187-203
Kern H, Schroder T, Kaulfuss M, Martin M, Kox WJ, Spies CD. Enoximone in contrast to dobutamine improves hepatosplanchnic function in fluid-optimized septic shock patients. Critical Care Medicine. 2001; 29:1519-1525
Santarpino G, Caroleo S, Onorati F, Dimastromatteo G, Abdalla K, Amantea B, et al. Inflammatory response to cardiopulmonary bypass with enoximone or steroids in patients undergoing myocardial revascularization: A preliminary report study. International Journal of Clinical Pharmacology and Therapeutics. 2009; 47:78-88
Pearse DB, Shimoda LA, Verin AD, Bogatcheva N, Moon C, Ronnett GV, et al. Effect of cGMP on lung microvascular endothelial barrier dysfunction following hydrogen peroxide. Endothelium. 2003; 10:309-317
Suttorp N, Weber U, Welsch T, Schudt C. Role of phosphodiesterases in the regulation of endothelial permeability in vitro. The Journal of Clinical Investigation. 1993; 91:1421-1428
Fuhrmann M, Jahn HU, Seybold J, Neurohr C, Barnes PJ, Hippenstiel S, et al. Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells. American Journal of Respiratory Cell and Molecular Biology. 1999; 20:292-302
Nagai K, Watanabe M, Seto M, Hisatsune A, Miyata T, Isohama Y. Nitric oxide decreases cell surface expression of aquaporin-5 and membrane water permeability in lung epithelial cells. Biochemical and Biophysical Research Communications. 2007; 354:579-584
Sidhaye V, Hoffert JD, King LS. cAMP has distinct acute and chronic effects on aquaporin-5 in lung epithelial cells. The Journal of Biological Chemistry. 2005; 280:3590-3596
Fanelli V, Puntorieri V, Assenzio B, Martin EL, Elia V, Bosco M, et al. Pulmonary-derived phosphoinositide 3-kinase gamma (PI3Kgamma) contributes to ventilator-induced lung injury and edema. Intensive Care Medicine. 2010; 36:1935-1945
Mokra D, Drgova A, Pullmann R Sr, Calkovska A. Selective phosphodiesterase 3 inhibitor olprinone attenuates meconium-induced oxidative lung injury. Pulmonary Pharmacology & Therapeutics. 2012; 25:216-222
Van der Mey M, Bommele KM, Boss H, Hatzelmann A, Van Slingerland M, Sterk GJ, et al. Synthesis and structure-activity relationships of cis-tetrahydrophthalazinone/pyridazinone hybrids: a novel series of potent dual PDE3/PDE4 inhibitory agents. Journal of Medicinal Chemistry. 2003; 46:2008-2016
Haddad JJ, Land SC, Tarnow-Mordi WO, Zembala M, Kowalczyk D, Lauterbach R. Immunopharmacological potential of selective phosphodiesterase inhibition. I. Differential regulation of lipopolysaccharide-mediated proinflammatory cytokine (interleukin-6 and tumor necrosis factor-alpha) biosynthesis in alveolar epithelial cells. The Journal of Pharmacology and Experimental Therapeutics. 2002; 300:559-566
Barnes PJ. Theophylline. American Journal of Respiratory and Critical Care Medicine. 2013; 188:901-906
Lambrecht BN, Hammad H. Asthma: The importance of dysregulated barrier immunity. European Journal of Immunology. 2013; 43:3125-3137
Lau HY, Kam MF. Inhibition of mast cell histamine release by specific phosphodiesterase inhibitors. Inflammation Research. 2005; 54(Suppl. 1):S05-S06
Milara J, Navarro A, Almudever P, Lluch J, Morcillo EJ, Cortijo J. Oxidative stress-induced glucocorticoid resistance is prevented by dual PDE3/PDE4 inhibition in human alveolar macrophages. Clinical and Experimental Allergy. 2011; 41:535-546
Gantner F, Schudt C, Wendel A, Hatzelmann A. Characterization of the phosphodiesterase (PDE) pattern of in vitro-generated human dendritic cells (DC) and the influence of PDE inhibitors on DC function. Pulmonary Pharmacology & Therapeutics. 1999; 12:377-386
Heystek HC, Thierry AC, Soulard P, Moulon C. Phosphodiesterase 4 inhibitors reduce human dendritic cell inflammatory cytokine production and Th1-polarizing capacity. International Immunology. 2003; 15:827-835
Gantner F, Tenor H, Gekeler V, Schudt C, Wendel A, Hatzelmann A. Phosphodiesterase profiles of highly purified human peripheral blood leukocyte populations from normal and atopic individuals: A comparative study. The Journal of Allergy and Clinical Immunology. 1997; 100:527-535
Barber R, Baillie GS, Bergmann R, Shepherd MC, Sepper R, Houslay MD, et al. Differential expression of PDE4 cAMP phosphodiesterase isoforms in inflammatory cells of smokers with COPD, smokers without COPD, and nonsmokers. American Journal of Physiology. Lung Cellular and Molecular Physiology. 2004; 287:L332-L343
Landells LJ, Spina D, Souness JE, O'Connor BJ, Page CP. A biochemical and functional assessment of monocyte phosphodiesterase activity in healthy and asthmatic subjects. Pulmonary Pharmacology & Therapeutics. 2000; 13:231-239
Giembycz MA, Corrigan CJ, Seybold J, Newton R, Barnes PJ. Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2. British Journal of Pharmacology. 1996; 118:1945-1958
Banner KH, Press NJ. Dual PDE3/4 inhibitors as therapeutic agents for chronic obstructive pulmonary disease. British Journal of Pharmacology. 2009; 157:892-906
Essayan DM, Kagey-Sobotka A, Lichtenstein LM, Huang SK. Differential regulation of human antigen-specific Th1 and Th2 lymphocyte responses by isozyme selective cyclic nucleotide phosphodiesterase inhibitors. The Journal of Pharmacology and Experimental Therapeutics. 1997; 282:505-512
Peter D, Jin SL, Conti M, Hatzelmann A, Zitt C. Differential expression and function of phosphodiesterase 4 (PDE4) subtypes in human primary CD4+ T cells: predominant role of PDE4D. Journal of Immunology. 2007; 178:4820-4831
Averill LE, Stein RL, Kammer GM. Control of human T-lymphocyte interleukin-2 production by a cAMP-dependent pathway. Cellular Immunology. 1988; 115:88-99
Bazhin AV, Kahnert S, Kimpfler S, Schadendorf D, Umansky V. Distinct metabolism of cyclic adenosine monophosphate in regulatory and helper CD4+ T cells. Molecular Immunology. 2010; 47:678-684
Gerlo S, Kooijman R, Beck IM, Kolmus K, Spooren A, Haegeman G. Cyclic AMP: A selective modulator of NF-kappaB action. Cellular and Molecular Life Sciences. 2011; 68:3823-3841
Rodriguez G, Ross JA, Nagy ZS, Kirken RA. Forskolin-inducible cAMP pathway negatively regulates T-cell proliferation by uncoupling the interleukin-2 receptor complex. The Journal of Biological Chemistry. 2013; 288:7137-7146
Feng G, Nadig SN, Backdahl L, Beck S, Francis RS, Schiopu A, et al. Functional regulatory T cells produced by inhibiting cyclic nucleotide phosphodiesterase type 3 prevent allograft rejection. Science Translational Medicine. 2011; 3:83ra40
Shih CH, Huang TJ, Chen CM, Lin YL, Ko WC. S-petasin, the main sesquiterpene of petasites formosanus, inhibits phosphodiesterase activity and suppresses ovalbumin-induced airway hyperresponsiveness. Evidence-based Complementary and Alternative Medicine. 2011; 2011:132374
Weston MC, Peachell PT. Regulation of human mast cell and basophil function by cAMP. General Pharmacology. 1998; 31:715-719
Rivington RN, Boulet LP, Cote J, Kreisman H, Small DI, Alexander M, et al. Efficacy of Uniphyl, salbutamol, and their combination in asthmatic patients on high-dose inhaled steroids. American Journal of Respiratory and Critical Care Medicine. 1995; 151:325-332
Yasui K, Agematsu K, Shinozaki K, Hokibara S, Nagumo H, Yamada S, et al. Effects of theophylline on human eosinophil functions: comparative study with neutrophil functions. Journal of Leukocyte Biology. 2000; 68:194-200
Yasui K, Hu B, Nakazawa T, Agematsu K, Komiyama A. Theophylline accelerates human granulocyte apoptosis not via phosphodiesterase inhibition. The Journal of Clinical Investigation. 1997; 100:1677-1684
Bethke T, Eschenhagen T, Klimkiewicz A, Kohl C, von der Leyen H, Mehl H, et al. Phosphodiesterase inhibition by enoximone in preparations from nonfailing and failing human hearts. Arzneimittel-Forschung. 1992; 42:437-445
Fujimura M, Kamio Y, Saito M, Hashimoto T, Matsuda T. Bronchodilator and bronchoprotective effects of cilostazol in humans in vivo. American Journal of Respiratory and Critical Care Medicine. 1995; 151:222-225
Herbert C, Hettiaratchi A, Webb DC, Thomas PS, Foster PS, Kumar RK. Suppression of cytokine expression by roflumilast and dexamethasone in a model of chronic asthma. Clinical and Experimental Allergy. 2008; 38:847-856
Fabbri LM, Calverley PMA, Izquierdo-Alonso JL, Bundschuh DS, Brose M, Martinez FJ, et al. Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: Two randomised clinical trials. Lancet. 2009; 374:695-703
Burgess JK, Oliver BG, Poniris MH, Ge Q, Boustany S, Cox N, et al. A phosphodiesterase 4 inhibitor inhibits matrix protein deposition in airways in vitro. The Journal of Allergy and Clinical Immunology. 2006; 118:649-657
Rabe KF. Update on roflumilast, a phosphodiesterase 4 inhibitor for the treatment of chronic obstructive pulmonary disease. British Journal of Pharmacology. 2011; 163:53-67
Kereiakes D, Chatterjee K, Parmley WW, Atherton B, Curran D, Kereiakes A, et al. Intravenous and oral Mdl-17043 (a new inotrope-vasodilator agent) in congestive heart-failure—Hemodynamic and clinical-evaluation in 38 patients. Journal of the American College of Cardiology. 1984; 4:884-889
Rubin SA, Tabak L. Mdl-17043—Short-term and long-term cardiopulmonary and clinical effects in patients with heart-failure. Journal of the American College of Cardiology. 1985; 5:1422-1427
Shah PK, Amin DK, Hulse S, Shellock F, Swan HJC. Inotropic therapy for refractory congestive heart-failure with oral fenoximone (Mdl-17,043)—Poor long-term results despite early hemodynamic and clinical improvement. Circulation. 1985; 71:326-331
Boswell-Smith V, Cazzola M, Page CP. Are phosphodiesterase 4 inhibitors just more theophylline? Journal of Allergy and Clinical Immunology. 2006; 117:1237-1243
Mokry J, Mokra D. Immunological aspects of phosphodiesterase inhibition in the respiratory system. Respiratory Physiology & Neurobiology. 2013; 187:11-17
Schudt C, Tenor H, Hatzelmann A. Pde isoenzymes as targets for antiasthma drugs. European Respiratory Journal. 1995; 8:1179-1183
- VM was supported by the NHLBI Intramural Research Program. Dr. Manganiello died on January 10, 2016