PDE Activity Research Articles

Zaprinast impairs spatial memory by increasing PDE5 expression in the rat hippocampus

In this work, we report the effect of post-training intraperitoneal administration of zaprinast on rat memory retention in the Morris water maze task that revealed a significant memory impairment at the intermediate dose of 10mg/kg. Zaprinast is capable of inhibiting both striatal and hippocampal PDE activity but to a different extent which is probably due to the different PDE isoforms expressed in these areas. To assess the possible involvement of cyclic nucleotides in rat memory impairment, we compared the effects obtained 30min after the zaprinast injection with respect to 24h after injection by measuring both cyclic nucleotide levels and PDE activity. As expected, 30min after the zaprinast administration, we observed an increase of cyclic nucleotides, which returned to a basal level within 24h, with the exception of the hippocampal cGMP which was significantly decreased at the dose of 10mg/kg of zaprinast. This increase in the hippocampal region is the result of a cGMP-specific PDE5 induction, confirmed by sildenafil inhibition, in agreement with literature data that demonstrate transcriptional regulation of PDE5 by cAMP/cGMP intracellular levels. Our results highlight the possible rebound effect of PDE inhibitors.

Role of Ser102 and Ser104 as regulators of cGMP hydrolysis by PDE5A.

BackgroundPhosphodiesterases (PDEs) cleave phosphodiester bonds in cyclic nucleotides and play diverse roles in cell biology. PDE5A is a cytoplasmic phosphodiesterase which specifically degrades cyclic guanosine monophosphate (cGMP), a cell signaling molecule that plays important roles in neuronal signaling and vascular smooth muscle contraction. Inhibition of PDE5A induces headache resembling migraine headaches.AimTo test the hypothesis that Ser102 and Ser104 in PDE5A and/or their phosphoserine derivatives 1) regulate the intracellular localization and/or activity of PDE5A, and 2) modulate the interaction between PDE5A and pharmaceutical reagents in clinical or pre-clinical use for migraine headaches and other types of vascular dysfunction.MethodsWild type PDE5A or PDE5A with substitution mutations (Ser102Ala, Ser104Ala or Ser102Ala/Ser104Ala) were overexpressed in SK-N-AS neuroblastoma cells as C-terminal fusions with green fluorescent protein. Transfected cells were treated with sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5A-GFP fusion proteins were localized in fixed cells by immunofluorescence and PDE activity was quantified in cell extracts by standard in vitro assay using [3H] cGMP.ResultsThe intracellular distribution of wild-type, single and double mutant PDE5A was similar and was not altered by exposure to sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5 activity was similar for wild type, Ser102Ala and Ser104Ala PDE5A, but activity of the Ser102Ala/Ser104Ala mutant was approximately two-fold higher than wild type. Double mutant Ser102Ala/Ser104Ala migrated as a single band on a native acrylamide gel, while wild-type and single mutant PDE5A migrated as a doublet.InterpretationSer102 and Ser104 may influence the conformational flexibility of PDE5A, which may in turn influence phosphorylation status, allosteric regulation by cGMP or other as yet unknown regulatory mechanisms for PDE5A. PDE5A activation could be important in reversal of migraine-like headache.

AB184. Effects of target gene expression on ex vivo differentiated mesenchymal stromal cells transfected by lent viral vector with PDE5 of short hairpin RNA

IntroductionIt has been shown that the major causes of erectile dysfunction (ED) associated with aging were via a diffuse and progressive corporal fibrosis to cavernous smooth muscle cells (CSMCs) in the penis. Previous studies have demonstrated that bone marrow-mesenchymal stromal cells (BM-MSCs) could possess the ability for self-renewal, multiline age differentiation and in vivo functional regeneration of damaged tissues. It was suggested that they be induced to differentiate into smooth muscle-like cells in vitro and have a potential source for stem-cell-based gene therapy of ED. Specific phosphodiesterases (PDE5) has been proved to play an important role in regulating penile erection to return to flaccidity by hydrolysis of cyclic guanosine monophosphate (cGMP) as a specific hydratase. Therefore, inhibiting specifically the catalytic activity of the target gene PDE5 has been indicated to promote the accumulation of cGMP and enhance erection. In recent years, the introduction of short hairpin RNA (shRNA) has proved to be a powerful tool for suppressing strongly gene specific expression through a process known as RNA interference. This study was to describe that stem cell-based replacement therapy with rat BM-MSCs by transfected the lentiviral vector carrying silencing target gene PDE5 of short hairpin RNA (PDE5-shRNA) could aid in the regeneration of penile smooth musculature and might help to benefit recovery of erectile function.PurposeTo investigate the expression of PDE5 in ex vivo differentiation of gene-modified BM-MSCs using the lentiviral vector containing silencing target gene PDE5 of shRNA.Methods and materialsSD rat bone marrow-derived MSCs were separated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent culture. The third passage rat BM-MSCs were obtained, and were identified by cell surface markers with flow cytometry (FCM). The lentiviral vector carrying silencing target gene PDE5 of shRNA was constructed and then transfected into the third passage rat BM-MSCs. The gene-modified rat BM-MSCs were induced to differentiate into smooth muscle-like cells exposed to VEGF and b-FGF median vitro. The proliferative ability of these cells was tested by cell counting kit 8 (CCK-8). Smooth muscle-like cells differentiation was assessed by immunofluorescence and then subjected to immunocytochemistry for specific markers of α-smooth muscle actin (α-SMA). Real-time quantitative PCR, immunohistochemical staining and Western blot analysis for PDE5 gene expression in differentiated smooth muscle-like cells were carried out.ResultsAfter the conducted lentiviral vector containing PDE5-shRNA was transfected into rat BM-MSCs, the expression of EGFP was detected at 24 hands it became the strongest at 72 h, which FCM showed the transfection efficiency were 91.3% at this time. The EGFP expression rates of rat BM-MSCs transfected with PDE5-shRNA at 3 d, 7 d, 14 d after transduction were 91.3%, 86.1%, and 82.7%, respectively. There was still visible green fluorescence at 28 d after transfection. The gene-modified rat BM-MSCs with PDE5-shRNA were successfully induced to differentiate into smooth muscle-like cells. Transduction of the lentiviral vector PDE5-shRNA into rat BM-MSCs led to down-regulation of PDE5. The expression of PDE5 was reduced 67.2% by PDE5-shRNA compared with the control lentiviral vector. The proliferative property of rat BM-MSCs did not significantly change among those transfected with PDE5-shRNA (P<0.001). The differentiated rat BM-MSCs were identified to smooth muscle-like cells by immunocytochemistry.ConclusionsThese results suggest that the lentiviral vector with gene-specific silencing PDE5 of shRNA could effectively transfect into rat BM-MSCs. They reveal that the gene-modified rat BM-MSCs with PDE5-shRNAcould be successfully induced to differentiate into smooth muscle-like cells in vitro and inhibit the expression of target gene PDE5.SupportThis work was supported by National Natural Science Foundation of China (81050026, 81370702).

Curcumin analogues inhibit phosphodiesterase-5 and dilate rat pulmonary arteries.

Phosphodiesterase (PDE)-5 inhibitors are useful as vasodilators for the treatment of pulmonary arterial hypertension. We aimed to study curcumin analogues for PDE5 inhibitory activity and vasorelaxation of rat pulmonary arteries. Three natural curcuminoids (1-3) and six synthetic analogues (4-9) were tested for PDE5 and PDE6 inhibitory activities using enzymatic radioassay. Their vasorelaxation was measured using freshly isolated segments of rat pulmonary artery and aorta. Curcuminoids (1-3) mildly inhibited PDE5 (half maximal inhibitory concentration (IC50 ) = 18 µm): the metamethoxyl of curcumin was important for PDE5 inhibition. But hydroxyl rearrangements, removing both methoxyls and one ketomethylene, yielded the potent 7 and 9 (IC50 = 4 µm) (compared with sildenafil, IC50 = 0.03 µm). Only 1, 3 and 4 were PDE5 selective over PDE6. Triazole-carboxylic addition provided water-solubility while preserving potency. All analogues possessed concentration-dependent vasorelaxant activity on pulmonary arteries (40% of maximal effective concentration (EC40 ) = 29-90 µm, maximum response = 60-90% at 300 µm), while compounds (1-8) were weakly acting in aorta (maximum response <40%). Only demethoxycurcumin (2) and analogues 5, 8, 9 had endothelium-dependent actions. Sildenafil was highly potent (EC40 = 0.04 µm) and highly endothelium dependent in pulmonary artery but weak on intact aorta (EC40 = 1.8 µm). Activity profiles suggest actions through additional cell pathways for promoting vasorelaxation. Curcumin analogues are potential leads for developing efficacious and selective PDE5 inhibitors and other pathologies of pulmonary hypertension.

Phosphodiesterase (PDE) inhibitor torbafylline (HWA 448) attenuates burn-induced rat skeletal muscle proteolysis through the PDE4/cAMP/EPAC/PI3K/Akt pathway

Treatment of rats after burn-injury with the cyclic AMP phosphodiesterase (PDE) inhibitor, torbafylline (also known as HWA 448) significantly reversed changes in rat skeletal muscle proteolysis, PDE4 activity, cAMP concentrations and mRNA expression of TNFα, IL-6, ubiquitin and E3 ligases. Torbafylline also attenuated muscle proteolysis during in vitro incubation, and this effect was blocked by the inhibitor Rp-cAMPS. Moreover, torbafylline significantly increased phospho-Akt levels, and normalized downregulated phospho-FOXO1 and phospho-4E-BP1 in muscle of burn rats. Similarly, torbafylline also normalized phosphorylation levels of Akt and its downstream elements in TNFα+IFNγ treated C2C12 myotubes. Torbafylline enhanced protein levels of exchange protein directly activated by cAMP (Epac) both in skeletal muscle of burn rats and in TNFα+IFNγ treated C2C12 myotubes. Pretreatment with a specific antagonist of PI3K or Epac significantly reversed the inhibitory effects of torbafylline on TNFα+IFNγ-induced MAFbx mRNA expression and protein breakdown in C2C12 myotubes. Torbafylline inhibits burn-induced muscle proteolysis by activating multiple pathways through PDE4/cAMP/Epac/PI3K/Akt.

Smooth muscle specific Rac1 deficiency induces hypertension by preventing p116RIP3-dependent RhoA inhibition.

BackgroundIncreasing evidence implicates overactivation of RhoA as a critical component of the pathogenesis of hypertension. Although a substantial body of work has established that Rac1 functions antagonize RhoA in a broad range of physiological processes, the role of Rac1 in the regulation of vascular tone and blood pressure is not fully elucidated.Methods and ResultsTo define the role of Rac1 in vivo in vascular smooth muscle cells (vSMC), we generated smooth muscle (SM)‐specific Rac1 knockout mice (SM‐Rac1‐KO) and performed radiotelemetric blood pressure recordings, contraction measurements in arterial rings, vSMC cultures and biochemical analyses. SM‐Rac1‐KO mice develop high systolic blood pressure sensitive to Rho kinase inhibition by fasudil. Arteries from SM‐Rac1‐KO mice are characterized by a defective NO‐dependent vasodilation and an overactivation of RhoA/Rho kinase signaling. We provide evidence that Rac1 deletion‐induced hypertension is due to an alteration of cGMP signaling resulting from the loss of Rac1‐mediated control of type 5 PDE activity. Consequently, cGMP‐dependent phosphorylation and binding of RhoA with its inhibitory partner, the phosphatase‐RhoA interacting protein (p116RIP3), are decreased.ConclusionsOur data reveal that the depletion of Rac1 in SMC decreases cGMP‐dependent p116RIP3/RhoA interaction and the subsequent inhibition of RhoA signaling. Thus, we unveil an in vivo role of Rac1 in arterial blood pressure regulation and a new pathway involving p116RIP3 that contributes to the antagonistic relationship between Rac1 and RhoA in vascular smooth muscle cells and their opposite roles in arterial tone and blood pressure.

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.

Synthesis of N-(3-arylprop-2-ynyl)substituted olanzapine derivatives as potential inhibitors of PDE4B

The linkage between dopamine D2 receptors and PDE activity via cAMP prompted us to design a series of novel N-(3-arylprop-2-ynyl)substituted olanzapine derivatives as potential inhibitors of PDE4B. The target compounds were conveniently prepared by using a simple and inexpensive method involving Pd/C-mediated CC bond forming reaction under Sonogashira conditions. A number of compounds were synthesized by using this strategy in good yields. Some of the compounds showed promising inhibition of PDE4B when tested in vitro that was supported by the docking studies.

Activation of the Pi3k/Akt Pathway and Modulation of Phosphodiesterase Activity via Membrane Progestin Receptor-Alpha (mPRalpha) Regulate Progestin-Initiated Sperm Hypermotility in Atlantic Croaker1

Rapid progestin stimulation of sperm motility is a widely observed phenomenon in vertebrates, but the mechanisms governing these effects are still poorly understood, especially in teleosts, amphibians, and birds, which do not express CatSper. Here we show that progestin-stimulated sperm hypermotility in a teleost, Atlantic croaker (Micropogonias undulatus), is initiated though membrane progestin receptor-alpha (mPRalpha, also known as Paqr7b) and involves activation of the Pi3k/Akt pathway and increased phosphodiesterase (Pde) activity. The specific mPRalpha agonist 10-ethenyl-19-norprogesterone (Org OD 02-0) mimicked the stimulatory actions of the endogenous progestin in this species, 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S), on sperm motility. Inhibition of Pi3k (1 nM Wortmannin; 25 μM LY294002) and Akt (25 μM ML-9) effectively abolished progestin-initiated sperm hypermotility. Surprisingly, treatment with the PDE inhibitors Cilostamide (100 nM) and Rolipram (1 μM) also blocked progestin stimulation of sperm motility. Whereas treatment with 20beta-S increased sperm Pde activity, pretreatment with Wortmannin eliminated this response to the progestin and also resulted in elevated cAMP levels, indicating that Pdes are at least partially under Pi3k/Akt control. The results suggest that mPRalpha-mediated progestin stimulation of sperm motility in croaker through Pi3k/Akt is dependent on maintenance of Pde activity and a reduction in internal cAMP concentrations. However, a previous study showed that progestin stimulation of sperm hypermotility in this species is also dependent on membrane adenylyl cyclase (Acy) activation and increased intrasperm cAMP concentrations. Collectively, these findings indicate that progestin-mediated hypermotility through mPRalpha in teleost sperm involves modulation of intracellular cAMP concentrations through multiple signaling pathways.

Endogenous hydrogen sulfide decreases Rho kinase and cGMP‐specific phosphodiesterase (PDE5) activity and induces relaxation of colonic smooth muscle (1110.3)

H2S, synthesized endogenously from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS), play a role in the regulation of gastrointestinal (GI) motility and secretion. Neither the expression of enzymes nor the mechanism of action of H2S in GI smooth muscle is fully known. Methods: Expression of CSE and CBS was analyzed by RT‐PCR and western blot. The effect of H2S on carbachol (CCh)‐induced Rho kinase activity and sustained contraction, and nitric oxide‐induced PDE5 activity, cGMP formation and muscle relaxation was examined using L‐cysteine (activator of CSE) or NaHS (H2S donor). Results: CSE, but not CBS was expressed in colonic smooth muscle cells of mouse, rabbit and human. CCh‐induced contraction in muscle strips and cells, and increased Rho kinase activity were inhibited by L‐cysteine and NaHS in a concentration‐dependent manner. Inhibition of Rho kinase activity and muscle contraction by L‐cysteine (but not NaHS) was blocked by CSE siRNA and CSE inhibitor, propargylglycine, respectively. L‐cysteine and NaHS also inhibited PDE5 activity, and augmented cGMP formation and muscle relaxation leading to increased (35%) colonic pellet propulsion. Conclusion: Activation of H2S‐producing CSE causes muscle relaxation via a dual mechanism involving inhibition of Rho kinase and PDE5 activity leading to downregulation of RhoA/Rho kinase pathway and upregulation of cGMP/PKG pathway.Grant Funding Source: DK‐28300

Upregulation of RhoA/Rho kinase pathway and downregulation of cGMP/PKG pathway in smooth muscle impair peristaltic reflex and pellet propulsion in the colon of diabetic mice (1110.5)

Abnormalities in extrinsic and intrinsic innervation and in the distribution of interstitial cells of Cajal are known to contribute to altered gut motility in diabetes. Smooth muscle dysfunction in relation to diabetes has not been fully explored. Aim. To test the hypothesis that upregulation of RhoA/Rho kinase pathway and downregulation of cGMP/PKG pathway contribute to increased muscle tone in diabetes. Methods. Colonic smooth muscle cells from type 1 (NOD) or type 2 diabetes (db/db) mice were used to measure i) RhoA and PDE5 expression; 2) ACh‐induced Rho kinase activity and muscle contraction; 3) NO‐induced PDE5 activity, cGMP levels and muscle relaxation. Colonic segments were used to measure peristaltic reflex and pellet propulsion. Results. Smooth muscle cells isolated from the colon of NOD and db/db mice showed an increase in RhoA and PDE5 expression, Rho kinase and PDE5 activity, and muscle contraction, and a decrease in cGMP levels and muscle relaxation relative to control mice. Consistent with an increase in RhoA and PDE5 expression, orad contraction was increased by 20%, caudad relaxation decreased by 60%, and pellet propulsion decreased by 35% in db/db mice relative to control. Discussion. In diabetic colon upregulation of the RhoA/Rho kinase pathway and downregulation of the cGMP/PKG pathway causes an increase in muscle contraction and decrease in relaxation leading to a decrease in pellet propulsion.

0097: Role of cAMP phosphodiesterases in the regulation of BK (Ca) channels in rat coronary arteries

3′-5′-cyclic adenosine monophosphate (cAMP) is an important mediator of vasorelaxation. In vascular smooth muscle, subcellular cAMP concentrations are mainly regulated by type 3 and 4 phosphodiesterases (PDE3 & PDE4). Large conductance Ca 2+ -activated K + channels (BK Ca ) channels are reported to mediate cAMP-induced relaxation. Here, we sought to clarify whether BK Ca channels are regulated by PDE activities in rat coronary arteries (CA) following β-adrenergic stimulation. CA were rapidly isolated from euthanized adult male Wistar rats, cut in 2 mm length rings and mounted on a wire myograph. Cumulative concentration-relaxation experiments were conducted on rings contracted with the thromboxane analogue U46619 (0.1–3 μM). The BK Ca inhibitor iberiotoxin (IBTX, 0.1 μM) did not alter pD 2 of isoproterenol (ISO) response. However, IBTX significantly reduced maximal relaxation induced by ISO, from 97±0.3% to 42±0.1% (N=10–12 animals, P<0.001). In rings without a functional endothelium, IBTX still significantly reduced ISO maximal response (36±3%, N=5, P<0.001) compared to vehicle (100±1%, N=6). Endothelium denudation of rCA rings slightly reduced ISO potency compared to intact rings, and this was observed either in the presence (N=6) or in the absence (N=5) of IBTX. In intact rings, inhibition of PDE3 using 1 μM cilostamide (Cil) and of PDE4 using 10 μM Ro20-1724 (Ro) increased ISO potency (N=9, P<0.01; N=6, P< 0.001, respectively, compared to vehicle: pD 2 =7.7±0.1, N=14). However, Cil and Ro were without effect in the presence of IBTX (N=6–9). These results show that, in rat CA, i) BK Ca channels are important effectors of ISO-induced relaxation; ii) BK Ca -dependent component of the response to ISO is not affected by endothelium removal; and iii) inhibition of PDE3 or PDE4 potentiates the response to ISO, an effect that requires active BK Ca channels.

Involvement of inducible nitric oxide synthase and dimethylarginine dimethylaminohydrolase in Nω‐Nitro‐L‐arginine methyl ester (L‐NAME)‐induced hypertension (LB676)

Chronic administration of L‐NAME in rats is an excellent model to study the induction and progression of a NO deficiency‐induced endothelial dysfunctional state. This study was performed to determine how different vascular beds compensate for the loss in nitric oxide. Male Wistar rats received L‐NAME (50mg/kg/day in drinking water) or no drug for 6 weeks. Aorta, pulmonary and renal arteries were isolated for determination of basal cGMP production, PDE5 activity, dimethylarginine dimethylaminohydrolase (DDAH) and hemeoxygenase (HO) activity. mRNA expression studies were done by real time PCR. Plasma IL‐1β level was measured by ELISA. Results: L‐NAME‐induced hypertension was associated with an increase in mean arterial pressure and plasma IL‐1β. The treatment had varied effects on mRNA expression levels of eNOS, soluble guanylyl cyclase, PDE5 and HO1 but showed an increased iNOS and decreased DDAH2 expression in all three tissues studied. Basal cGMP levels were decreased in all tissues, but PDE5 activity was increased only in renal and pulmonary artery. DDAH activity was significantly reduced, whereas HO activity was significantly increased in all the tissues. This study showed that different vascular beds do not react similarly to chronic NO inhibition and that vascular inflammation triggered by an increase in iNOS and indirectly by a decrease in DDAH activity might be the causative factors for the progression of vascular dysfunction in this model.

0310 Assay development and high-throughput screening to identify PDE2A activators to treat heart failure

Chronic activation of β-adrenergic (β-AR) pathway causes excessive accumulation of cAMP in cardiomyocytes and participates in pathological cardiac remodeling leading to heart failure (HF). Cyclic nucleotide phosphodiesterases (PDEs) are responsible for the degradation of cAMP and cGMP in subcellular compartments. Among the different PDE families, PDE2 is unique in being activated ˜;5-fold by cGMP, thus mediating a negative cross talk between cGMP and cAMP signaling. Recently, we found that PDE2A activity is increased in human HF and this may represent a protective mechanism limiting â-AR cardiotoxicity in chronic HF. Our therapeutic strategy is to target PDE2 with specific activators to treat HF at the early stages. A PDE2 activity assay has been developed, first using a purified PDE2 from bovine adrenal glands, then using a commercial recombinant protein (full-length human PDE2A). We miniaturized a bioluminescent activity assay in 384 well plates, which was validated for High-Throughput Screening (HTS) with an excellent Z-factor (>0.7) on the Ciblot HTS platform. Moreover, an in silico screening based on the binding of molecules on the GAF-B domain (regulatory domain) of PDE2 has been performed on a large library of molecules. As a counter screen, a PDE4 assay has been employed to eliminate nonspecific modulators. Ranking of hits is based on activation efficacy in comparison to 5 μM cGMP, M EC50, Emax and cytotoxicity following chemical resynthesis in a SAR (Structure Activity Relationship) strategy. Several complementary assays are currently under development to confirm the effect of the activators in a Hit to Lead process (cellular assays, cAMP ELISA, co-cristallisation, Biacore). This challenging project is supported by LabEx LERMIT within its new cardiovascular target program

Cyclic GMP catabolism up-regulation in MRL/lpr lupus-prone mice is associated with organ remodeling

Production of high titer of antibodies against nuclear components is a hallmark of systemic lupus erythematosus, an autoimmune disease characterized by the progressive chronic inflammation of multiple joints and organs. Organ damage and dysfunction such as renal failure are typical clinical features in lupus. Cell hypermetabolism and hypertrophy can accelerate organ dysfunction. In this study we focus on a specific murine model of lupus, the MRL/lpr strain, and investigated the role of cyclic guanosine monophosphate (cGMP) catabolism in organ remodeling of main target tissues (kidney, spleen and liver) in comparison with age-matched control mice. In MRL/lpr-prone mice, the cGMP-phosphodiesterase (PDE) activities were significantly increased in the kidney (3-fold, P<0.001), spleen (2-fold, P<0.001) and liver (1.6-fold, P<0.05). These raised activity levels were paralleled by both an increased activity of PDE1 in the kidney (associated with nephromegaly) and in the liver, and PDE2 in the spleen of lupus-prone mice. The up-regulation of PDE1 and PDE2 activities were associated with a decrease in intracellular cGMP levels. This underlines an alteration of cGMP-PDE signaling in the kidney, spleen and liver targeting different PDEs according to organs. In good agreement with these findings, a single intravenous administration to MRL/lpr mice of nimodipine (PDE1 inhibitor) but not of EHNA (PDE2 inhibitor) was able to significantly lower peripheral hypercellularity (P=0.0401), a characteristic feature of this strain of lupus-prone mice. Collectively, our findings are important for generating personalized strategies to prevent certain forms of the lupus disease as well as for understanding the role of PDEs and cGMP in the pathophysiology of lupus.

Excess adenosine A2B receptor signaling contributes to priapism through HIF‐1α mediated reduction of PDE5 gene expression

Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A(2B) adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA(-/-) and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.

Correction: NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity

Correction: NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity

NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.

Differential regulation of C-type natriuretic peptide-induced cGMP and functional responses by PDE2 and PDE3 in failing myocardium

Recently, we showed C-type natriuretic peptide (CNP)-induced negative inotropic (NIR) and positive lusitropic response (LR) in failing rat heart. We wanted to study whether, and if so, how phosphodiesterases (PDEs) regulate CNP-induced cyclic 3',5'-guanosine monophosphate (cGMP) elevation and functional responses. Inotropic and lusitropic responses were measured in left ventricular muscle strips and cyclic nucleotide levels, PDE activity and phospholamban (PLB) and troponin I (TnI) phosphorylation were measured in ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. CNP-mediated increase in global cGMP was mainly regulated by PDE2, as reflected by a marked amplification of the cGMP increase during PDE2 inhibition and by a high PDE2 activity in cardiomyocytes. PDE3 inhibition, on the other hand, caused no significant cGMP increase by CNP. The functional consequences did not correspond to the changes of cGMP. PDE3 inhibition increased the potency of the CNP-induced NIR and LR, while PDE2 inhibition desensitized the CNP-induced NIR, but not LR. A role for PDE2 on the maximal LR and PDE5 on the maximal NIR to CNP was revealed in the presence of PDE3 inhibition. CNP increased PLB phosphorylation about 25- to 30-fold and tended to increase TnI phosphorylation about twofold. As a whole, CNP-induced functional responses were only modestly regulated by PDEs compared to the cAMP-mediated functional responses to β1-adrenoceptor stimulation, which are highly regulated by PDEs. There is a mismatch between the CNP-induced cGMP increase and functional responses. Global cGMP levels are mainly regulated by PDE2 after CNP stimulation, whereas the functional responses are modestly regulated by both PDE2 and PDE3, indicating cGMP compartmentation by PDEs affecting CNP-induced responses in failing hearts.

Effects of inactivated Bordetella pertussis on phosphodiesterase in the lung of ovalbumin sensitized and challenged rats.

This paper indicated that inactivated Bordetella pertussis (iBp) can enhance the lung airway hyperreactivity of the rats sensitized and challenged with OVA. The mechanisms were involved in the upregulation of cAMP-PDE activity and PDE4A, PDE4D, and PDE3 gene expression in the lungs. But only PDE4 activity was different between the OVA and OVA+iBp groups, and PDE4D expression was significantly increased in iBp rats alone. So, our data suggested that cosensitization with OVA and iBp affects lung airway reactivity by modulating the lung cAMP-PDE activity and PDE4D gene expression.