shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa

In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca²⁺) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP‐gated calcium sodium channels (CNG) to increase Na⁺ and Ca²⁺ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na⁺ and Ca²⁺ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca²⁺ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca²⁺ to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b^H620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b^H620Q mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.     

Inactivation of Pde8b enhances memory,motor performance,and protects against age‐induced motor coordination decay

Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor‐coordination, and have an attenuation of age‐induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value .     

Phosphodiesterase‐1b deletion confers depression‐like behavioral resistance separate from stress‐related effects in mice

Phosphodiesterase‐1b (Pde1b) is highly expressed in striatum, dentate gyrus, CA3 and substantia nigra. In a new Floxed Pde1b × Cre^CMV global knockout (KO) mouse model, we show an immobility‐resistance phenotype that recapitulates that found in constitutive Pde1b KO mice. We use this new mouse model to show that the resistance to acute stress‐induced depression‐like phenotype is not the product of changes in locomotor activity or reactivity to other stressors (learned helplessness, novelty suppressed feeding or dexamethasone suppression), and is not associated with anhedonia using the sucrose preference test. Using tamoxifen inducible Cre, we show that the immobility‐resistant phenotype depends on the age of induction. The effect is present when Pde1b is Reduced from conception, P0 or P32, but not if reduced as adults (P60). We also mapped regional brain expression of PDE1B protein and of the Cre driver. These data add to the suggestion that PDE1B may be a target for drug development with therapeutic potential in depression alone or in combination with existing antidepressants.     

A mouse homolog of dunce,a gene important for learning and memory in drosophila,is preferentially expressed in olfactory receptor neurons

The dunce (dnc) gene in Drosophila codes for a cyclic adenosine monophosphate-specific phosphodiesterase (PDE). Flies with a mutation at this locus exhibit severe deficits in learning and memory. We have begun to analyze the neural distribution of mammalian homologs of dnc in the mouse. Surprisingly, in situ hybridization and northern blotting using a probe specific for one of the four mammalian dnc homologs (mPDE2) reveals high levels of expression in the olfactory neuroepithelium. Anti mPDE2 antibody confirms that this PDE protein is abundant in the axons and dendrites of the olfactory receptor neurons but is conspicuosly absent from the cilia, where the initial events in olfactory signal transduction occur. Lower levels of mPDE2 were also detected throughout the brain and in the testis. These findings suggest an important modulatory role for mPDE2 in mammalian olfaction. © 1995 John Wiley & Sons, Inc.     

Inhibition of phosphodiestrase 9 induces cGMP accumulation and apoptosis in human breast cancer cell lines, MCF-7 and MDA-MB-468

Objectives

Phosphodiesterase 9 (PDE9) is a major isoform of phosphodiesterase hydrolysing cGMP and plays a key role in proliferation of cells, their differentiation and apoptosis, via intracellular cGMP signalling. The study described here was designed to investigate expression, activity and apoptotic effect of PDE9 on human breast cancer cell lines, MCF‐7 and MDA‐MB‐468.

Materials and methods

Activity and expression of PDE9 were examined using colorimetric cyclic nucleotide phosphodiesterase assay and real‐time RT‐PCR methods respectively; cGMP concentration was also measured. MTT viability test, annexin V‐FITC staining, Hoechst 33258 staining and caspase3 activity assay were used to detect apoptosis.

Results

Treatment of both cell lines with BAY 73‐6691 lead to reduction in PDE9 mRNA expression, PDE9 cGMP‐hydrolytic activity and elevation of the intracellular cGMP response. BAY 73‐6691 significantly reduced cell proliferation in a dose‐ and time‐dependent manner and caused marked increase in apoptosis through caspase3 activation.

Conclusion

Our results revealed that BAY 73‐6691 induced apoptosis in these breast cancer cell lines through the cGMP pathway. These data suggest that BAY 73‐6691 could be utilized as an agent in treatment of breast cancer.     

A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle

Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M₂AChR pharmacological profile. Therefore, a novel Ca²⁺/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58?kDa) being the 58?kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [³H]cGMP and [³H]cAMP exhibiting a higher affinity as K_m (μM) for cGMP than cAMP but being close values with V_max cAMP/cGMP ratio of 1.5. The co-factor Mg²⁺ showed similar K_(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC₅₀ of 4.9 and 4.6?μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K_(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M₂AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.     

ANP and CNP activate CFTR expressed in Xenopus laevis oocytes by direct activation of PKA

Abstract

Context: Acting through different receptors, natriuretic peptides (atrial natriuretic peptide [ANP], brain type natriuretic peptide [BNP] and C-type natriuretic peptide [CNP]) increase intracellular cGMP, which then stimulates different pathways that activate fluid secretion. Objective: We used two-electrode voltage clamping to define the dominant pathway that is employed when natriuretic peptides activate cystic fibrosis transmembrane conductance regulator (CFTR) in the Xenopus oocyte expression system. Natriuretic peptides could activate CFTR by 1) cGMP cross-activation of protein kinase A (PKA), 2) cGMP activation of cGMP-dependent protein kinase II, 3) cGMP inhibition of phosphodiesterase type III (PDE3), or 4) direct activation of CFTR. Materials and Methods: cRNA-microinjected Xenopus laevis oocytes were perfused with diverse compounds that examined these pathways of natriuretic peptide signaling. Results and Discussion: ANP stimulated the shark CFTR (sCFTR)-mediated chloride conductance and this activation was inhibited by H-89, a specific inhibitor of PKA. After co-expression of the CNP receptor (NPR-B), sCFTR became stimulatable by CNP and was similarly inhibited by H-89, pointing to cross-activation of PKA. 8-pCPT-cGMP, a relatively cGKII-selective cGMP, failed to stimulate sCFTR. Another membrane-permeable and non-hydrolyzable analog of cGMP, 8-Br-cGMP, stimulated CFTR only at millimolar concentrations, consistent with cross-activation of PKA. The PDE inhibitors EHNA, rolipram, cilostamide, and amrinone did not significantly increase chloride conductance, arguing against a significant role for PDE2, PDE3 and PDE4 signaling in the oocyte. Sildenafil, a PDE5 inhibitor, caused a partial activation of sCFTR channels and this effect was again inhibited by H-89. Conclusion: From these experiments we conclude that in the Xenopus oocyte system, natriuretic peptides, 8-Br-cGMP, and PDE5 inhibitors activate CFTR by cross-activation of PKA.     

Function of the asparagine 74 residue of the inhibitory γ-subunit of retinal rod cGMP-phophodiesterase (PDE) in vivo

The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αβ. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g^tm1 mice. This transgenic line was crossed with Pde6g^tm1/Pde6g^tm1 mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αβ may be more dynamic and context-dependent than was replicated in vitro.     

Profiling of cAMP and cGMP phosphodiesterases in isolated ventricular cardiomyocytes from human hearts: comparison with rat and guinea pig

AimsPhosphodiesterases (PDEs) are key enzymes controlling cAMP and cGMP levels and spatial distribution within cardiomyocytes. Despite the clinical importance of several classes of PDE inhibitor there has not been a complete characterization of the PDE profile within the human cardiomyocyte, and no attempt to assess which species might best be used to model this for drug evaluation in heart disease.Main methodsVentricular cardiomyocytes were isolated from failing human hearts of patients with various etiologies of disease, and from rat and guinea pig hearts. Expression of PDE isoforms was determined using RT-PCR. cAMP- and cGMP-PDE hydrolytic activity was determined by scintillation proximity assay, before and after treatment with PDE inhibitors for PDEs 1, 2, 3, 4, 5 and 7. Functional effects of cAMP PDEi were determined on the contraction of single human, rat and guinea pig cardiomyocytes.Key findingsThe presence and activity of PDE5 were confirmed in ventricular cardiomyocytes from failing and hypertrophied human heart, as well as PDE3, with ventricle-specific results for PDE4 and a surprisingly large contribution from PDE1 for hydrolysis of both cAMP and cGMP. The total PDE activity of human cardiomyocytes, and the profile of inhibition by PDE1, 3, 4, and 5 inhibitors, was modelled well in guinea pig but not rat cardiomyocytes.SignificanceOur results provide the first full characterisation of human cardiomyocyte PDE isoforms, and suggest that guinea pig myocytes provide a better model than rat for PDE levels and activity.     

Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h^−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h^−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h^−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.     

Cyclic Nucleotides and Neuroblastoma Differentiation

We have shown that intracellular cGMP levels increase during retinoic acid‐ and mycophenolic acid‐induced neuroblastoma differentiation and that a 6 days treatment with 1 mM dbcGMP lead LAN5 cell to elaborate a network of neuritic processes suggesting an involvement of cGMP in neuroblastoma differentiation. We have also investigated the effects of some specific inhibitors of phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) on human neuroblastoma (LAN5 and SHEP) growth and differentiation. After six days of incubation in the presence of each specific inhibitor at 10 × IC₅₀ levels a cytostatic and differentiating effect was only observed with the PDE5 inhibitors Zaprinast and MY‐5445. The cytostatic effect of these compounds increased increasing their concentrations far above their IC₅₀ levels for PDE5, suggesting that these compounds could act by interfering with other molecular events than direct cGMP‐PDE inhibition. No appreciable effect was observed using Dipyridamole, another specific PDE5 inhibitor.     

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.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Effect of the ILE86TER mutation in the γ subunit of cGMP phosphodiesterase (PDE6) on rod photoreceptor signaling

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g^tm1/Pde6g^tm1 mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile⁸⁶ and Ile⁸⁷ residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100 μM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile⁸⁶ and Ile⁸⁷ residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.     

Biochemical and pharmacological characterization of cyclic nucleotide phosphodiesterase in airway epithelium

According to their respective elution order, specificity for cAMP and cGMP, their sensitivity to calmodulin, and their modulation by cGMP and rolipram, four cyclic nucleotide phosphodiesterases (PDE) were separated from the cytosol: PDE I (calmodulin-sensitive), PDE II (stimulated by cGMP, PDE IV (cGMP specific-PDE and inhibited by rolipram) and PDE V (cGMP specific). PDE IV (K_m=1.4 M) was competitively inhibited rolipram (K_i=1.2 M) whereas PDE V (K_m=0.83 M) was competitively inhibited by zaprinast in the molar range (K_i=0.12 M). Moreover the microsomal fraction contained three PDE isoforms: PDE II, PDE III (inhibited by cGMP or indolidan) and PDE IV. These results show that cAMP degradation in cytosolic and membrane fractions is modulated by cGMP and selectively inhibited by rolipram and, in addition, by indolidan in membrane fractions. (Mol Cell Biochem140: 171–175, 1994)     

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′,5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K_I = 43.4 ± 7.2 μM and k_cart = 0.007 ± 0.0006 min⁻¹). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K_d = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD⁺, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.     

The Phosphodiesterase 9 Inhibitor PF‐04449613 Promotes Dendritic Spine Formation and Performance Improvement after Motor Learning

The cyclic nucleotide cGMP is an intracellular second messenger with important roles in neuronal functions and animals' behaviors. The phosphodiesterases (PDEs) are a family of enzymes that hydrolyze the second messengers cGMP and cAMP. Inhibition of phosphodiesterase 9 (PDE9), a main isoform of PDEs hydrolyzing cGMP, has been shown to improve learning and memory as well as cognitive function in rodents. However, the role of PDE9 in regulating neuronal structure and function in vivo remains unclear. Here we used in vivo two‐photon microscopy to investigate the effect of a selective PDE9 inhibitor PF‐04449613 on the activity and plasticity of dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex. We found that administration of PF‐04449613 increased calcium activity of dendrites and dendritic spines of layer V pyramidal neurons in mice under resting and running conditions. Chronic treatment of PF‐04449613 over weeks increased dendritic spine formation and elimination under basal conditions. Furthermore, PF‐04449613 treatment over 1–7 days increased the formation and survival of new spines as well as performance improvement after rotarod motor training. Taken together, our studies suggest that elevating the level of cGMP with the PDE9 inhibitor PF‐04449613 increases synaptic calcium activity and learning‐dependent synaptic plasticity, thereby contributing to performance improvement after learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2018     

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.