Phosphodiesterase 7 inhibition preserves dopaminergic neurons in cellular and rodent models of Parkinson disease

Background

Phosphodiesterase 7 plays a major role in down-regulation of protein kinase A activity by hydrolyzing cAMP in many cell types. This cyclic nucleotide plays a key role in signal transduction in a wide variety of cellular responses. In the brain, cAMP has been implicated in learning, memory processes and other brain functions.

Methodology/Principal Findings

Here we show a novel function of phosphodiesterase 7 inhibition on nigrostriatal dopaminergic neuronal death. We found that S14, a heterocyclic small molecule inhibitor of phosphodiesterase 7, conferred significant neuronal protection against different insults both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. S14 treatment also reduced microglial activation, protected dopaminergic neurons and improved motor function in the lipopolysaccharide rat model of Parkinson disease. Finally, S14 neuroprotective effects were reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase A.

Conclusions/Significance

Our findings demonstrate that phosphodiesterase 7 inhibition can protect dopaminergic neurons against different insults, and they provide support for the therapeutic potential of phosphodiesterase 7 inhibitors in the treatment of neurodegenerative disorders, particularly Parkinson disease.     

β-Adrenergic cAMP Signals Are Predominantly Regulated by Phosphodiesterase Type 4 in Cultured Adult Rat Aortic Smooth Muscle Cells

Background

We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs).

Methodology/Principal Findings

The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3  =  PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β₁- and β₂-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β₁- and β₂-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation.

Conclusion/Significance

Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.     

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

Background

Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.

Methodology/Principal Findings

We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem ^−/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.

Conclusion/Significance

Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.     

β2-Agonist induced cAMP is decreased in asthmatic airway smooth muscle due to increased PDE4D

Background and Objective

Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known.

Objective

To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism.

Methods

We examined β₂-adrenergic (β₂AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined.

Results

In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM.

Conclusion

Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.     

Olprinone Attenuates the Acute Inflammatory Response and Apoptosis after Spinal Cord Trauma in Mice

Background

Olprinone hydrochloride is a newly developed compound that selectively inhibits PDE type III and is characterized by several properties, including positive inotropic effects, peripheral vasodilatory effects, and a bronchodilator effect. In clinical settings, olprinone is commonly used to treat congestive cardiac failure, due to its inotropic and vasodilating effects. The mechanism of these cardiac effects is attributed to increased cellular concentrations of cAMP. The aim of the present study was to evaluate the pharmacological action of olprinone on the secondary damage in experimental spinal cord injury (SCI) in mice.

Methodology/Principal Findings

Traumatic SCI is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should be preventable, no effective treatment options currently exist for patients with SCI. Spinal cord trauma was induced in mice by the application of vascular clips (force of 24 g) to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, apoptosis, and locomotor disturbance. Olprinone treatment (0.2 mg/kg, i.p.) 1 and 6 h after the SCI significantly reduced: (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, (4) pro-inflammatory cytokines, (5) NF-κB expression, (6) p-ERK1/2 and p38 expression and (7) apoptosis (TUNEL staining, FAS ligand, Bax and Bcl-2 expression). Moreover, olprinone significantly ameliorated the recovery of hind-limb function (evaluated by motor recovery score).

Conclusions/Significance

Taken together, our results clearly demonstrate that olprinone treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

Background

Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2''-deoxy-2''-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo.

Methods

Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs.

Results

In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used.

Conclusion

These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

Delayed Treatment with Systemic (S)-Roscovitine Provides Neuroprotection and Inhibits In Vivo CDK5 Activity Increase in Animal Stroke Models

Background

Although quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment.

Methodology/Principal Findings

In the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms.

Conclusions/Significance

Altogether, our data bring new evidences for the further development of pharmacological CDK inhibitors in stroke therapy.     

Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models

Background

Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.

Methodology/Principal Findings

We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [¹⁴C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [¹⁴C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).

Conclusions/Significance

These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.     

Sildenafil reduces insulin-resistance in human endothelial cells

Background

The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro.

Methodology/Principal Findings

Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine.

Conclusions/Significance

These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients.     

Pharmacotherapy in pulmonary arterial hypertension: a systematic review and meta-analysis

Background

Previous meta-analyses of treatments for pulmonary arterial hypertension (PAH) have not shown mortality benefit from any individual class of medication.

Methods

MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials were searched from inception through November 2009 for randomized trials that evaluated any pharmacotherapy in the treatment of PAH. Reference lists from included articles and recent review articles were also searched. Analysis included randomized placebo controlled trials of at least eight weeks duration and studies comparing intravenous medication to an unblinded control group.

Results

1541 unique studies were identified and twenty-four articles with 3758 patients were included in the meta-analysis. Studies were reviewed and data extracted regarding study characteristics and outcomes. Data was pooled for three classes of medication: prostanoids, endothelin-receptor antagonists (ERAs), and phosphodiesterase type 5 (PDE5) inhibitors. Pooled relative risks (RRs) and 95% confidence intervals (CIs) were calculated for mortality, 6-minute walk distance, dyspnea scores, hemodynamic parameters, and adverse effects. Mortality in the control arms was a combined 4.2% over the mean study length of 14.9 weeks. There was significant mortality benefit with prostanoid treatment (RR 0.49, CI 0.29 to 0.82), particularly comparing intravenous agents to control (RR 0.30, CI 0.14 to 0.63). Mortality benefit was not observed for ERAs (RR 0.58, CI 0.21 to 1.60) or PDE5 inhibitors (RR 0.30, CI 0.08 to 1.08). All three classes of medication improved other clinical and hemodynamic endpoints. Adverse effects that were increased in treatment arms include jaw pain, diarrhea, peripheral edema, headache, and nausea in prostanoids; and visual disturbance, dyspepsia, flushing, headache, and limb pain in PDE5 inhibitors. No adverse events were significantly associated with ERA treatment.

Conclusions

Treatment of PAH with prostanoids reduces mortality and improves multiple other clinical and hemodynamic outcomes. ERAs and PDE5 inhibitors improve clinical and hemodynamic outcomes, but have no proven effect on mortality. The long-term effects of all PAH treatment requires further study.     

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated,but not rhinovirus infected,primary human airway smooth muscle

Background

Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE₄) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations.

Methods

In the present study we investigated whether the PDE₄ inhibitor piclamilast (10^-6 M) could alter RV or viral mimetic (5 μg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E₂ and cAMP production were assayed by ELISA and RV replication was assayed by viral titration.

Results

We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines.

Conclusion

By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE₄ inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.     

Characterization of novel StAR (steroidogenic acute regulatory protein) mutations causing non-classic lipoid adrenal hyperplasia

Context

Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH).

Objective

StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported.

Design

To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature.

Setting

Collaboration between the University Children''s Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d''Hebron, Autonomous University, Barcelona, Spain.

Patients

Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age.

Results

StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol.

Conclusions

StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.     

Protective effect of pioglitazone on morphine-induced neuroinflammation in the rat lumbar spinal cord

Background

Morphine-induced tolerance is associated with the spinal neuroinflammation. The aim of this study was to explore the effects of oral administration of the pioglitazone, the peroxisome proliferator activated receptor gamma (PPAR-γ) agonist, on the morphine-induced neuroinflammation in the lumbar region of the male Wistar rat spinal cord.

Results

Co-administration of the pioglitazone with morphine not only attenuated morphine-induced tolerance, but also prevented the up-regulation of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1beta, and interleukin 6) and nuclear factor-kappa B activity. Administration of the GW-9662 antagonized the above mentioned effects of the pioglitazone.

Conclusions

It is concluded that oral administration of the pioglitazone attenuates morphine-induced tolerance and the neuroinflammation in the lumbar region of the rat spinal cord. This action of the pioglitazone may be, at least in part, due to an interaction with the spinal pro-inflammatory cytokine expression and the nuclear factor-kappa B activity.     

Role of Ser102 and Ser104 as Regulators of cGMP Hydrolysis by PDE5A

Background

Phosphodiesterases (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.

Aim

To 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.

Methods

Wild 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.

Results

The 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.

Interpretation

Ser102 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.     

Impaired neonatal macrophage phagocytosis is not explained by overproduction of prostaglandin E2

Background

Neonates and young infants manifest increased susceptibility to bacterial, viral and fungal lung infections. Previous work has identified a role for eicosanoids in mediating host defense functions of macrophages. This study examines the relationship between alveolar macrophage (AM) host defense and production of lipid mediators during the neonatal period compared to adult AMs.

Methods

AMs were harvested from young (day 7 and day 14) and adult (~10 week) rats. The functionality of these cells was assessed by examining their ability to phagocytose opsonized targets, produce cytokines, eicosanoids and intracellular cAMP measured by enzyme immunoassays, and gene expression of proteins, enzymes and receptors essential for eicosanoid generation and phagocytosis measured by real time RT-PCR.

Results

AMs from young animals (day 7 and 14) were defective in their ability to phagocytose opsonized targets and produce tumor necrosis factor (TNF)- α. In addition, young AMs produce more prostaglandin (PG) E₂, a suppressor of host defense, and less leukotriene (LT) B₄, a promoter of host defense. Young AMs express higher levels of enzymes responsible for the production of PGE₂ and LTB₄; however, there was no change in the expression of E prostanoid (EP) receptors or LT receptors. Despite the similar EP profiles, young AMs are more responsive to PGE₂ as evidenced by their increased production of the important second messenger, cyclic AMP. In addition, young AMs express higher levels of PDE3B and lower levels of PDE4C compared to adult AMs. However, even though the young AMs produced a skewed eicosanoid profile, neither the inhibition of PGE₂ by aspirin nor the addition of exogenous LTB₄ rescued the defective opsonized phagocytosis. Examination of a receptor responsible for mediating opsonized phagocytosis showed a significant decrease in the gene expression levels of the Fcgamma receptor in young (day 7) AMs compared to adult AMs.

Conclusion

These results suggest that elevated production of PGE₂ and decreased production of LTB₄ do not contribute to impaired opsonized macrophage phagocytosis and highlight an important difference between young and adult AMs.     

A biphasic and brain-region selective down-regulation of cyclic adenosine monophosphate concentrations supports object recognition in the rat

Background

We aimed to further understand the relationship between cAMP concentration and mnesic performance.

Methods and Findings

Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h).

Conclusions

Our results strongly suggest that a “pre-sample” early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay.