Role Of Phosphodiesterases Research Articles

Role of Phosphodiesterase 1 in the Regulation of Real-Time cGMP Levels and Contractility in Adult Mouse Cardiomyocytes.

In mouse cardiomyocytes, the expression of two subfamilies of the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase 1 (PDE1)-PDE1A and PDE1C-has been reported. PDE1C was found to be the major subfamily in the human heart. It is a dual substrate PDE and can hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Previously, it has been reported that the PDE1 inhibitor ITI-214 shows positive inotropic effects in heart failure patients which were largely attributed to the cAMP-dependent protein kinase (PKA) signaling. However, the role of PDE1 in the regulation of cardiac cGMP has not been directly addressed. Here, we studied the effect of PDE1 inhibition on cGMP levels in adult mouse ventricular cardiomyocytes using a highly sensitive fluorescent biosensor based on Förster resonance energy transfer (FRET). Live-cell imaging in paced and resting cardiomyocytes showed an increase in cGMP after PDE1 inhibition with ITI-214. Furthermore, PDE1 inhibition and PDE1A knockdown amplified the cGMP-FRET responses to the nitric oxide (NO)-donor sodium nitroprusside (SNP) but not to the C-type natriuretic peptide (CNP), indicating a specific role of PDE1 in the regulation of the NO-sensitive guanylyl cyclase (NO-GC)-regulated cGMP microdomain. ITI-214, in combination with CNP or SNP, showed a positive lusitropic effect, improving the relaxation of isolated myocytes. Immunoblot analysis revealed increased phospholamban (PLN) phosphorylation at Ser-16 in cells treated with a combination of SNP and PDE1 inhibitor but not with SNP alone. Our findings reveal a previously unreported role of PDE1 in the regulation of the NO-GC/cGMP microdomain and mouse ventricular myocyte contractility. Since PDE1 serves as a cGMP degrading PDE in cardiomyocytes and has the highest hydrolytic activities, it can be expected that PDE1 inhibition might be beneficial in combination with cGMP-elevating drugs for the treatment of cardiac diseases.

β‐Asarone attenuates streptozotocin‐induced experimental dementia in rats: Possible role of phosphodiesterases and cyclic nucleotide signaling

AbstractBackgroundIn recent years, extract of Acorus calamus shown to inhibit phosphodiesterases (PDE) and β‐asarone has been reported to be a major active constituent. The purpose of the present study was to ascertain PDE inhibitory potential of β‐asarone and to explore its therapeutic potential in sporadic Alzheimer’s type dementia (SAD). In the current study, PDE inhibitory potential of β‐asarone was determined using in vitro assay system.MethodStreptozotocin (STZ) was infused intracerebroventrically [bilaterally (3 mg/kg) ICV] to induce SAD in rats. Spatial and non‐spatial memory was evaluated using Morris water maze and object recognition task in rats.Rats were treated with β‐asarone (12.5, 25 and 50 mg/kg) from day 10 to 21 following 1st STZ infusion. On day 22 rats were sacrificed and the cortical and hippocampal brain regions were used to identify biochemical alterationsResultSTZ infused rats showed significant learning and memory deficit which was linked with increase in oxidative stress (malondialdehyde and nitrite), compromised antioxidant defense (reduced glutathione), mitochondrial dysfunction (deficit in complex I and III activity) and pro‐inflammatory cytokine levels. Significant decrease in both cAMP and cGMP levels were also observed in STZ‐infused rats. β‐Asarone dose dependently attenuated STZ‐induced cognitive deficit, biochemical alterations and restored cyclic nucleotide levels.ConclusionThe observed results show important role of PDE inhibition and cyclic nucleotide signaling in the beneficial effects of β‐asarone and suggest therapeutic potential of β‐asarone in the management of cognitive disorders.

Pde5 Inhibition Reduced Blood Pressure and Alleviated Target Organ Damage in Chronic Intermittent Hypoxia.

The role of phosphodiesterase 5 (Pde5) in obstructive sleep apnea-induced damage remains unclear. Our study aimed to investigate the role of Pde5 in the chronic intermittent hypoxia (CIH) model. C57BL/6J wild-type (WT) mice (n = 48) and Pde5 knockout (Pde5 -/- ) mice (n = 24) were randomly assigned to CIH group and room air group. After 6 weeks, some WT mice (n = 24) in CIH group were given sildenafil or saline gavage for another 4 weeks. Blood pressure was regularly measured during the experiment. Echocardiography was used to estimate cardiac function. We collected organs from each group of mice and measured their physical indicators. Histochemical staining was used to explore the size of cardiomyocyte and fibrosis area of various organs. Cyclic guanosine monophosphate and malondialdehyde concentrations in serum were measured by ELISA assay. Compared with the RA-treated group, the 6-week CIH resulted in a significant increase in blood pressure, altered heart structure, and reduced serum cyclic guanosine monophosphate in WT mice. Pde5 -/- mice and sildenafil intragastric administration significantly reduced systolic blood pressure in CIH condition and attenuated the damage of target organs. In CIH model, we found that the cardiomyocyte size and fibrosis area of heart and kidney significantly reduced in Pde5 -/- groups. Besides, endogenous and exogenous inhibition of Pde5 reduced malondialdehyde level and inflammatory and oxidative stress markers expression in CIH condition. In this study, we found that Pde5 inhibition could reduce blood pressure and alleviate target organ damage in the CIH model, which may be mediated through the oxidative stress pathway.

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling

Proliferation of smooth muscle cells, oxidative stress, and pulmonary vasoconstriction resulting from intermittent hypoxia (IH) facilitate pulmonary hypertension (PH) in patients with obstructive sleep apnea. The role of Phosphodiesterase 4 B (PDE4B) in PH has not yet been established. Herein, we investigated whether PDE4B inhibition ameliorates experimental PH by modulating cAMP signaling. We performed an integrative analysis of PDE4B expression in Gene Expression Omnibus datasets, experimental IH-induced rat PH samples, and IH-induced pulmonary arterial smooth muscle cells (PASMCs). PDE4B expression was modulated using siRNA in vitro and a specific adeno-associated virus serotype 1 in vivo. In the databases of mouse models of IH-induced and sustained hypoxia-induced PH and in a rat model of six weeks of IH, the expression of PDE4B was up-regulated. Inhibition of PDE4B attenuated IH-induced pulmonary vascular remodeling and right ventricular hypertrophy. Our results also showed that PDE4B deficiency inhibited IH-induced proliferation of PASMCs with less mitochondrial reactive oxygen species and mitochondrial damage. Meanwhile, IH induced an increase in ATF4, which positively regulated the expression of PDE4B through transcription, and inhibition of ATF4 exerted effects similar to those of PDE4B inhibition. Mechanistically, downregulating the expression of PDE4B resulted in the activation of the cAMP/PKA/p-CREB/PGC-1α pathway in PASMCs after IH. Taken together, our present study provides evidence that inhibition of PDE4B attenuates IH-induced PH by regulating cAMP signaling.

Exercise During Pregnancy Improves PDE-BKCa Coupling In Cerebral Arteries In Offspring Of Spontaneously Hypertensive Rats

In numerous cardiovascular diseases, Phosphodiesterases (PDEs) mediate ion channel pathological compensation in vascular smooth muscle cells (VSMCs). Our previous studies showed that exercise during pregnancy improves ion channel function in VSMCs of offspring in spontaneously hypertensive rats (SHR). The hypothesis that the pathological compensation of large-conductance Ca2+-activated K+ (BKCa) channels mediated by PDE in cerebral arteries of SHR is tested here. It is further demonstrated that exercise during pregnancy enhances the functional coupling of PDE to BKCa channels to improve cerebral arterial function in the offspring of SHR. PURPOSE: To investigate the effect of exercise during pregnancy on BKCa channel and the role of PDE in regulating BKCa function of cerebral arteries in the offspring of SHR. Methods: Pregnant SHR and Wistar-Kyoto rats (WKY) were separated into a sedentary group (p-WKY-SED, p-SHR-SED) and an exercise group (p-WKY-EX, p-SHR-EX) at random. Exercise group were subjected to swimming (60 min/day, 6 day/week, gestation day 1-19), and the BKCa channel function of the cerebral arteries from the 3-month-old (3 M) offspring were evaluated. Results: Exercise during pregnancy significantly decreased the resting mean arterial blood pressure at 3 M in the offspring of SHR (SHR-EX, 153.2 ± 7.5 vs. SHR-SED, 164.6 ± 3.6 mmHg, p < 0.05). The BKCa current density in p-SHR-SED offspring (49.8 ± 7.3 pA/pF) was markedly higher than that in p-WKY-SED (36.8 ± 6.1 pA/pF, p < 0.05). Exercise during pregnancy significantly decreased BKCa channel currents in p-SHR-EX (33.4 ± 11.2 pA/pF, p < 0.05 vs. p-SHR-SED). After application of PDE inhibitor (cilostamide and Ro-20-1724), the percentage of current enhancement was higher in p-WKY-SED (44.3 ± 15.1%) than in p-SHR-SED (13.5 ± 12.1%, p < 0.05), whereas exercise during pregnancy significantly increased it in p-SHR-EX (45.2 ± 3.1%, p < 0.05 vs. p-SHR-SED). Conclusions: These data suggest that the loss of PDE-BKCa coupling mediates pathological compensation of BKCa channels in the cerebral arteries of SHR offspring. Exercise during pregnancy enhances the coupling, and delays the development of hypertension. Supported by the National Natural Science Foundation of China 32071174.

TLR4/NF-κB/TNFα and cAMP/SIRT1 signaling cascade involved in mediating the dose-dependent effect of cilostazol in ovarian ischemia reperfusion-induced injury

Background One of the most dangerous gynecological emergencies is ovarian ischemia that commonly occurs during surgical manipulation or presence of ovarian masses. Objectives finding new therapies to prevent the associated harmful effects of ischemia/reperfusion-induced damage is still a critical need. For the first time, we aimed to evaluate the possible role of phosphodiesterase (PDE) 3 A inhibitor (PDEI), cilostazol (CLZ) in the treatment of ovarian ischemia reperfusion induced damage (OIR). Methods Rats were divided into five groups; sham, OIR group; CLZ (5, 10, 20 mg/kg) was given orally with induced OIR. Different biochemical parameters were detected such as total anti-oxidant capacity (TAC), reduced glutathione (GSH), malondialdehyde (MDA), cyclic adenosine monophosphate (cAMP), sirtuin1 (SIRT1), toll like receptor 4 (TLR4), nuclear factor kappa b (NF-κB) and tumor necrosis factor alpha (TNFα). In addition, histopathological features, ovarian weight changes and casapse3 immunoexpression were detected. Results Data revealed significant increase in ovarian weight changes, MDA, TLR4, TNFα, NF-κB and caspase 3 expressions in OIR induced group. Moreover, OIR group had histopathological features of ovarian damage with depletion of cAMP, SIRT1, TAC and GSH. Conclusion CLZ could ameliorate OIR-induced damage due to PDE inhibition, anti-oxidant, anti-inflammatory and anti-apoptotic properties with modulation of TLR4/NF-κB/TNFα and cAMP/SIRT1 signaling pathways.

Phosphodiesterases and Compartmentation of cAMP and cGMP Signaling in Regulation of Cardiac Contractility in Normal and Failing Hearts.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.

PDE inhibition in distinct cell types to reclaim the balance of synaptic plasticity.

Synapses are the functional units of the brain. They form specific contact points that drive neuronal communication and are highly plastic in their strength, density, and shape. A carefully orchestrated balance between synaptogenesis and synaptic pruning, i.e., the elimination of weak or redundant synapses, ensures adequate synaptic density. An imbalance between these two processes lies at the basis of multiple neuropathologies. Recent evidence has highlighted the importance of glia-neuron interactions in the synaptic unit, emphasized by glial phagocytosis of synapses and local excretion of inflammatory mediators. These findings warrant a closer look into the molecular basis of cell-signaling pathways in the different brain cells that are related to synaptic plasticity. In neurons, intracellular second messengers, such as cyclic guanosine or adenosine monophosphate (cGMP and cAMP, respectively), are known mediators of synaptic homeostasis and plasticity. Increased levels of these second messengers in glial cells slow down inflammation and neurodegenerative processes. These multi-faceted effects provide the opportunity to counteract excessive synapse loss by targeting cGMP and cAMP pathways in multiple cell types. Phosphodiesterases (PDEs) are specialized degraders of these second messengers, rendering them attractive targets to combat the detrimental effects of neurological disorders. Cellular and subcellular compartmentalization of the specific isoforms of PDEs leads to divergent downstream effects for these enzymes in the various central nervous system resident cell types. This review provides a detailed overview on the role of PDEs and their inhibition in the context of glia-neuron interactions in different neuropathologies characterized by synapse loss. In doing so, it provides a framework to support future research towards finding combinational therapy for specific neuropathologies.

Role of Phosphodiesterase in the Biology and Pathology of Diabetes.

Glucose metabolism is the initiator of a large number of molecular secretory processes in β cells. Cyclic nucleotides as a second messenger are the main physiological regulators of these processes and are functionally divided into compartments in pancreatic cells. Their intracellular concentration is limited by hydrolysis led by one or more phosphodiesterase (PDE) isoenzymes. Literature data confirmed multiple expressions of PDEs subtypes, but the specific roles of each in pancreatic β-cell function, particularly in humans, are still unclear. Isoforms present in the pancreas are also found in various tissues of the body. Normoglycemia and its strict control are supported by the appropriate release of insulin from the pancreas and the action of insulin in peripheral tissues, including processes related to homeostasis, the regulation of which is based on the PDE- cyclic AMP (cAMP) signaling pathway. The challenge in developing a therapeutic solution based on GSIS (glucose-stimulated insulin secretion) enhancers targeted at PDEs is the selective inhibition of their activity only within β cells. Undeniably, PDEs inhibitors have therapeutic potential, but some of them are burdened with certain adverse effects. Therefore, the chance to use knowledge in this field for diabetes treatment has been postulated for a long time.

Role of phosphodiesterases in the development of takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype in our former study, but the pathogenesis of TTS is still not completely understood. In this study our aim was to prove the hypothesis of an altered phosphodiesterase (PDE)-dependent 3',5'-cyclic adenosine monophosphate (cAMP)-signaling in TTS in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods and results We generated functional TTS-iPSC-CMs and treated them with catecholamines to mimic a TTS-phenotype. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used Förster resonance energy transfer (FRET) based cAMP sensors, which are specifically located in the cytosol or at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA) micro domain. We demonstrated that β-adrenergic receptor (β-AR) stimulations resulted in stronger cytosolic FRET responses in TTS-CMs compared to controls. In contrast, no differences of cAMP level were observed in the SERCA-PLN micro domain between TTS- and control-iPSC-CMs. To analyze the interplay of β-AR signaling and specific PDE contribution to the cAMP signaling in TTS, specific PDE-inhibitors were used. We were able to show in the cytosol that after β-AR stimulation, the strong effects of the PDE4 family of control cells were significantly decreased in diseased TTS CMs, which is in line with previously described reduced PDE4 activity in failing mouse hearts. In contrast, the contribution of PDE3 to cytoplasmic cAMP degradation was increased in TTS (Figure 1 A). This is in line with increased PDE3A and down-regulated PDE4D protein expression in TTS-iPSC-CMs compared to control cells. Analysis of PDE-dependent cAMP level in the SERCA micro domain show also a significantly reduced PDE4 activity. But the dynamic cytosolic PDE contribution of PDE2 and PDE3 after catecholamine treatment in TTS is lost in SERCA micro domain (Figure1B). Conclusion Our data showed for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. We demonstrated an isozym shift from PDE4 in control to PDE3 and PDE2 in TTS and identified PDE4 as an important player in the β-adrenergic cAMP signaling in TTS. Therefore, PDE4 activators may be a possible new therapeutic target option in the treatment of TTS. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK

Role of Phosphodiesterase 7 (PDE7) in T Cell Activity. Effects of Selective PDE7 Inhibitors and Dual PDE4/7 Inhibitors on T Cell Functions.

Phosphodiesterase 7 (PDE7), a cAMP-specific PDE family, insensitive to rolipram, is present in many immune cells, including T lymphocytes. Two genes of PDE7 have been identified: PDE7A and PDE7B with three or four splice variants, respectively. Both PDE7A and PDE7B are expressed in T cells, and the predominant splice variant in these cells is PDE7A1. PDE7 is one of several PDE families that terminates biological functions of cAMP—a major regulating intracellular factor. However, the precise role of PDE7 in T cell activation and function is still ambiguous. Some authors reported its crucial role in T cell activation, while according to other studies PDE7 activity was not pivotal to T cells. Several studies showed that inhibition of PDE7 by its selective or dual PDE4/7 inhibitors suppresses T cell activity, and consequently T-mediated immune response. Taken together, it seems quite likely that simultaneous inhibition of PDE4 and PDE7 by dual PDE4/7 inhibitors or a combination of selective PDE4 and PDE7 remains the most interesting therapeutic target for the treatment of some immune-related disorders, such as autoimmune diseases, or selected respiratory diseases. An interesting direction of future studies could also be using a combination of selective PDE7 and PDE3 inhibitors.

Commentary: Phosphodiesterase 4 inhibitors as potential adjunct treatment targeting the cytokine storm in COVID-19

The most severe presentation of COVID-19 is characterized by a hyperinflammatory state attributed to the massive pro-inflammatory cytokine release, called “cytokine storm”. Several specific anti-inflammatory/immunosuppressive agents are being evaluated by ongoing clinical trials; however, there is currently insufficient evidence for their efficacy and safety in COVID-19 treatment. Given the role of phosphodiesterase 4 (PDE) 4 and cyclic adenosine monophosphate in the inflammatory response, we hypothesize that selective PDE4 inhibition may attenuate the cytokine storm in COVID-19, through the upstream inhibition of pro-inflammatory molecules, particularly TNF-α, and the regulation of the pro-inflammatory/anti-inflammatory balance. Conversely, other anti-cytokine agents lead to the downstream inhibition of specific targets, such as IL-1, IL-6 or TNF-α, and may not be efficient in blocking the cytokine storm, once it has been triggered. Due to their mechanism of action targeting an early stage of the inflammatory response and ameliorating lung inflammation, we believe that selective PDE4 inhibitors may represent a promising treatment option for the early phase of COVID-19 pneumonia before the cytokine storm and severe multiorgan dysfunction take place. Furthermore, PDE4 inhibitors present several advantages including an excellent safety profile; the oral route of administration; the convenient dosing; and beneficial metabolic properties. Interestingly, obesity and diabetes mellitus type 2 have been reported to be risk factors for the severity of COVID-19. Therefore, randomized clinical trials of PDE4 inhibitors are necessary to explore their potential therapeutic effect as an adjunct to supportive measures and other therapeutic regiments.

The Role of Phosphodiesterase (Pde) 3B in the Inflammatory Response of Macrophages to LPS

Abstract Pde3B hydrolyzes both cAMP and cGMP, important second messengers in a variety of cellular processes. Pde3B has been shown to regulate NLRP3 inflammasome activity in adipose tissue. To understand whether Pde3B plays a role in the macrophage response to bacterial infection, we derived macrophages from the bone marrow of wildtype and Pde3B knockout mice. Macrophages were then stimulated with LPS for 6 h or left untreated, and total RNA was prepared for RNA-seq analyses. Our data indicate that Pde3B expression was inhibited by LPS in macrophages. Knockout of Pde3B resulted in significant changes in the expression of a large group of genes: 427 genes were up-regulated by at least 2-fold while 428 genes were down-regulated by more than 50% in LPS stimulated macrophages as the result of Pde3B deficiency. Gene profiling indicates that a number of biological processes are affected by Pde3B knockout, including cellular ion/cation homeostasis, immune system processes, and chemotaxis. Interestingly, Pde3B deficiency disturbed the expression of several chemokines: Cxcl-1, -2, and -3 expression were decreased by 96, 60, and 75%, respectively. In contrast, Ccl-4, -7, -12, -22, Cxcl-9 and -l11 expression were enhanced 1.8, 1.5, 1.9, 2, 3, and 3-fold, respectively. Differential expression of dual specificity phosphatase (Dusp)-1, -2, -3, -4, -5, -6, -10, -11, and -16 were also seen between LPS-stimulated wildtype and Pde3B-deficient macrophages. Since the Dusps are important regulators of MAP kinases that control the expression of chemokines, alterations in the expression of Dusps provides a plausible explanation for the changes in chemokine expression. Our studies suggest that Pde3B may regulate chemokine expression via modulating MAP kinases.

P2869Role of PDE8 in cAMP dynamics in human atrial fibrillation

Abstract Background Cardiac arrhythmias, such as atrial fibrillation (AF), are often related to remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca2+ handling mechanisms. For instance, decreased L-type calcium current (ICa,L) density but upregulated RyR2 are major hallmarks of AF. These inhomogeneous AF-associated changes of protein phosphorylation point to a local regulation of PKA activity within these intracellular compartments. Local cAMP compartmentation and the role of phosphodiesterase (PDEs) have ben extensively studied in ventricular myocytes from animals. However, only a few studies have evaluated the contribution of PDEs to the pathophysiology of AF and the reason for the persistent AF-associated hypophosphorylation of the L-type calcium channel (LTCC) is currently unknown. The aim of this study was to investigate whether a change in the expression level of PDE8 in human atrium may affects cAMP nearby LTCC promoting the reduction of the ICa,L observed in persistent AF. Methods Atrial myocytes were isolated from tissue of 47 patients in sinus rhythm (SR) and with AF. Cells were then transfect with an adenovirus (Epac1-camps or pm-Epac1-camps) in order to express the (cytosolic or membrane, respectively) FRET-based cAMP sensor and cultured during 48 hours. Föster-resonance energy transfer (FRET) was used to measure cAMP in 232 isolated human atrial myocytes. Ro-20-1724 (10 μM), Cilostamide (1 μM) and PF-04957325 (30 nM) and IBMX (100 μM) were used as PDE4, PDE3, PDE8 and non-selective phosphodiesterases (PDEs) inhibitor respectively. Results Effects of PDE4 and especially PDE3 inhibition on cytosolic [cAMP] are reduced in AF. Pharmacological PDE8 inhibition induces only a small increase in basal intracellular [cAMP] in AF but it showed a big synergic effect when PDE4 was inhibit at the same time. By contrast, PDE8 inhibition dramatically increased basal [cAMP] in the subsarcolemmal compartment in AF while PDE3 or PDE4 inhibition had a smaller effect that didn't change between SR and AF. Conclusions PDE8 controls basal cytosolic cAMP levels in human atrial myocytes from patients with persistent AF while PDE3 effects tends to be reduced in these patients. Furthermore, PDE8 is the main PDE in controlling cAMP levels at the membrane in persistent AF. Thus, our study may provide a clue for the reported reduction of the ICa,L in persistent AF.

P2863Regional specific remodeling of cGMP pathway in human atrial fibrillation

Abstract Background Atrial fibrillation (AF) is accompanied by a profound remodeling of membrane receptors and alterations in cyclic nucleotides-dependent regulation of Ca2+-handling. Thus, while basal ryanodine receptors activity is upregulated, L-type calcium current (ICa,L) density is diminish in AF, due to local microdomain-specific cAMP dynamics. The same seems true for cGMP regulation in AF. In AF cGMP-mediated increase in ICa,L is blunted but NO-mediated attenuation of β-adrenoceptors stimulation-mediated increase is preserved. However, although the role of cGMP in controling atrial function and pathophysiology is controversial, no study has been ever performed in human myocytes to measure cGMP directly. Methods We isolated myocytes from the right and/or left atrium of 27 patients in sinus rhythm (SR), and with AF. Cells were then transfected with adenovirus to express the cytosolic FRET-based cGMP sensor red-cGES-DE5 and cultured for 48 hours. Förster resonance energy transfer (FRET) was used to measure cGMP in 61 living human atrial myocytes. We stimulated cells with the C-type natriuretic peptide CNP (100 nM and 1 μM), and the non-selective phosphodiesterases (PDEs) inhibitor IBMX (100 μM). Additionally, PDE specific inhibitors for PDE2 (Bay 60–7550, 100 nM) and PDE3 (Cilostamide, 10 μM) as well as inhibitor of the soluble guanylyl cyclase (ODQ, 50 μM) were used. We also measured PDE2 and PDE3 mRNA levels in atrial tissue samples from both groups of patients using RT-qPCR. Results We could show that stimulation with CNP increased cGMP levels in human atrial myocytes. However, in myocytes from patients with AF global cGMP responses to CNP and to IBMX was reduced compared to SR. Additionally, there was a difference in response to CNP and IBMX in patients with AF between the right and the left atria. Whereas in the right atria IBMX could further increase cGMP levels in the cell, in the left atria leaded to a reduction in cGMP levels. RT-qPCR showed a tendency of PDE3 to be reduced in AF. On the other hand, PDE2A gene expression was upregulated in the left atria. Conclusions We have shown that PDEs contributes cGMP signaling in the human atria and that they are involved in atrial pathophysiology. Now our data clearly show differences in cGMP regulation in cardiomyocytes isolated from left and right atrium from patients in atrial fibrillation and sinus rhythm. We observe a major role of PDEs, regulating cGMP pathway promoted by the reduced responses in AF, especially PDE2 in the left atria.

Early administration of phosphodiesterase 5 inhibitors after induction of diabetes in a rat model may prevent erectile dysfunction.

The possible role of phosphodiesterase 5 inhibitors (PDE5Is) in prevention of negative effect of diabetes mellitus (DM) on erectile function is not well settled. To investigate the effect of early administration of vardenafil on erectile function, cavernosal structure, and genes expression in a rat model of DM. This experimental study was carried out atSuez Canal University's research laboratory. This study was conducted on a total of 60 adult male Albino Wistar rats, aged 60-80days and weighing an average of 200g. Rats were equally divided into six groups of 10 rats each: Group I (sham); Group II (DM with no treatment); Groups III, IV, V, and VI received vardenafil started at day 1, week 4, week 8, and week 12 after induction of DM, respectively. Functional study assessment of all groups was performed before euthanization, and then tissues were harvested for histopathological, ultrastructural, and molecular examinations. There was a significant difference of intracavernosal pressure between early (94±2.18) and late (40.5±1.94) treatment groups (p=0.011). Histopathological and ultrastructural changes of DM with no treatment and late treatment groups showed distorted cavernous architecture and extensive fibrosis. There was significant difference of smooth muscle to collagen ratio between early and late treatment groups (p=0.035). There was significant upregulation of nNOS(p=0.021) and iNOS (p=0.047) in early vs. late treatment group. The difference was insignificant in eNOS (p=0.386) or TGF-β1(p=0.149). Early treated rats with vardenafil had preserved erection and normal cavernosal structure, ultrastructure and gene expression of iNOS, nNOS, eNOS, and TGF-β1. Quantification of gene expression would improve our knowledge regarding cytokines expression and molecular background of DM-associated ED. Clinical application of this result may encourage early administration of PDE5I to prevent deleterious effects of DM on erectile function in newly diagnosed DM patients with probable uncontrolled blood glucose.

Knockout of adenylyl cyclase isoform 5 or 6 differentially modifies the β1-adrenoceptor-mediated inotropic response

Although only β2-adrenergic receptors (βAR) dually couple with stimulatory G protein (Gs) and inhibitory G protein (Gi), inactivation of Gi enhances both β1AR and β2AR responsiveness. We hypothesize that Gi restrains spontaneous adenylyl cyclase (AC) activity independent of receptor activation. Subcellular localization of the AC5/6 subtypes varies contributing to the compartmentation of βAR signaling. The primary objectives were to determine: (1) if β1AR-mediated inotropic responses were dependent upon either AC5 or AC6; (2) if intrinsic Gi inhibition is AC subtype selective and (3) the role of phosphodiesterases (PDE) 3/4 to regulate β1AR responsiveness. β1AR-mediated increases in contractile force and cAMP accumulation in cardiomyocytes were measured from wild type, AC5 and AC6 knockout (KO) mice, with or without pertussis toxin (PTX) pretreatment to inactivate Gi and/or after selective inhibition of PDEs 3/4. Noradrenaline potency at β1ARs was increased in AC6 KO. PDE4 inhibition increased noradrenaline potency in wild type and AC5 KO, but not AC6 KO. PTX increased noradrenaline potency only in wild type but increased the maximal β1AR response in all mouse strains. PDE3 inhibition increased noradrenaline potency only in AC5 KO that was treated prior with PTX. β1AR-evoked cAMP accumulation was increased more by PDE4 inhibition than PDE3 inhibition in wild type and AC5 KO that was amplified by Gi inhibition. These data indicate that β1AR-mediated inotropic responses are not dependent upon either AC5 or AC6 alone. Inactivation of Gi enhanced β1AR-mediated inotropic responses despite not coupling to Gi, consistent with Gi exerting a tonic receptor independent inhibition upon AC5/6. PDE4 seems the primary regulator of β1AR signaling through AC6 in wild type. AC6 KO results in a reorganization of β1AR compartmentation characterized by signaling through AC5 regulated by Gi, PDE3 and PDE4 that maintains normal contractile function.

Role of Phosphodiesterase 5 (PDE5) in Breast Cancer Stroma: Implications for Targeted Therapy

Breast cancers are heterogeneous tissues comprised of multiple components, including tumor and microenvironment cells. Cancer‐associated fibroblasts (CAFs) are an important cell type in tumor‐associated stroma with multiple roles in tumor initiation and promotion. While the significance of CAFs is well recognized, the treatment of CAFs in clinical practice has not yet been established. Our previous studies have shown that the overexpression of phosphodiesterase (PDE) 5, a metallohydrolase which catalyzes the hydrolytic breakdown of cGMP, enhances the invasive potential of breast cancer cells and reduces survival in breast cancer patients. To extend these observations, we propose to assess the function of PDE5 in tumor microenvironment, to further highlight the potential benefit of targeting PDE5.Our results evidenced, for the first time, PDE5 mRNA and protein expression in CAFs isolated from biopsies of primary human breast tumors. PDE5 expression is increased in human breast cancer stroma compared with normal breast stroma and was correlated to a shorter overall survival in patients. Treatment with PDE5 inhibitors (i.e. sildenafil and tadalafil) significantly inhibited CAF proliferation, motility and invasiveness. To better examine the significance of PDE5 expression in stromal fibroblasts, PDE5 was stably integrated into mouse embryonic fibroblasts (MEFs) and cell proliferation, motility and invasion were investigated. PDE5 transformed MEFs towards a cancer‐associated fibroblast phenotype, as evidenced by increased expression of CAF markers along with enhanced proliferative, migratory and invasive capabilities. Coculture experiments showed that proliferation and motility of human breast cancer cells was increased by PDE5‐overexpressing MEFs compared to vector‐expressing MEFs, suggesting that stromal PDE5 contributes to heterotypic communications within breast cancer. An enriched production of the chemokine CXCL16 was revealed in PDE5‐expressing clones, as evidenced by cytokine array and confirmed by realtime PCR, suggesting a role for this molecule in mediating the tumor‐promoting effects of PDE5 expression in breast stroma. In line with these findings, exposure of CAFs to PDE5 inhibitors was associated with a reduced expression of CXCL16. More importantly, expression of CXCL16 in breast cancer stroma showed a strong correlation with PDE5 as well as with a poor patient survival.In conclusion, PDE5 is overexpressed in breast cancer stroma and enhances the tumor‐stimulatory activities of fibroblasts, highlighting this enzyme as a novel prognostic candidate and an attractive target for future therapy in breast cancers, having already addressed the delivery, stability, and toxicity issues of PDE5 inhibitors in other diseases.Support or Funding InformationMy First AIRC Grant (MFAG) #16899 to I. BaroneThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of phosphodiesterase 4 in excessive daytime sleepiness in Parkinson's disease

IntroductionPreclinical studies suggest a link between cAMP/PKA signalling, phosphodiesterase 4 (PDE4) expression and excessive daytime sleepiness (EDS). Here, we investigated in vivo the association between PDE4 expression and EDS in Parkinson's disease (PD) patients using [11C]rolipram PET and MR imaging. MethodsEighteen participants, 12 PD and 6 healthy controls, underwent one [11C]rolipram PET and a multi-modal MRI scan. Probabilistic tractography was performed on subjects’ diffusion data to functionally parcellate the striatum according with projections to limbic cortical areas. The severity of EDS was assessed using the Epworth Sleepiness Scale (ESS). To assess PDE4 expression in PD patients with EDS, the PD cohort was divided according to the presence (n = 5) or absence (n = 7) of EDS, defined using validated cut-off of score ≥10 on the ESS as score ≥10 on the ESS. ResultsPD patients with EDS showed significantly increased [11C]rolipram volume of distribution (VT) in the caudate (P = 0.029), hypothalamus (P = 0.013), hippocampus (P = 0.036) and limbic striatum (P = 0.030) compared to patients without EDS. Furthermore, higher ESS scores correlated with increased [11C]rolipram VT in the caudate (r = 0.77; P = 0.003), hypothalamus (r = 0.84; P = 0.001), hippocampus (r = 0.81; P = 0.001) and limbic subdivisions of the striatum (r = 0.80; P = 0.003). ConclusionOur findings translate into humans preclinical data indicating that EDS is associated with elevated PDE4 in regions regulating sleep. The severity of EDS in PD was associated with elevated PDE4 expression; thus, suggesting a role of PDE4 in the pathophysiology of EDS in PD.