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.

Regulation of adrenal steroidogenesis by the high-affinity phosphodiesterase 8 family.

The main function of cyclic AMP phosphodiesterases (PDEs) is to degrade cAMP, a ubiquitous second messenger. Therefore, PDEs can function as prime regulators of cAMP/PKA-dependent processes such as steroidogenesis. Until recently, the roles of the PDE8 family have been largely unexplored, presumably due to the lack of a selective inhibitor. This review focuses on recent reports about the regulatory roles of the PDE8 family in adrenal steroidogenesis, as well as the inhibitory properties and specificity of a new PDE8-selective inhibitor, PF-04957325. We also describe a method of measuring urinary corticosterone levels in vivo as a minimally invasive way of monitoring the stress level in a mouse.

Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain.

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.

Upregulation of phosphodiesterase 1A1 expression is associated with the development of nitrate tolerance.

BACKGROUND: The efficacy of nitroglycerin (NTG) as a vasodilator is limited by tolerance, which develops shortly after treatment begins. In vascular smooth muscle cells (VSMCs), NTG is denitrated to form nitric oxide (NO), which activates guanylyl cyclase and generates cGMP. cGMP plays a key role in nitrate-induced vasodilation by reducing intracellular Ca(2+) concentration. Therefore, one possible mechanism for development of nitrate tolerance would be increased activity of the cGMP phosphodiesterase (PDE), which decreases cGMP levels. METHODS AND RESULTS: To test this hypothesis, rats were made tolerant by continuous infusion of NTG for 3 days (10 microgram kg(-1). min(-1) SC) with an osmotic pump. Analysis of PDE activities showed an increased function of Ca(2+)/calmodulin (CaM)-stimulated PDE (PDE1A1), which preferentially hydrolyzes cGMP after NTG treatment. Western blot analysis for the Ca(2+)/CaM-stimulated PDE revealed that PDE1A1 was increased 2.3-fold in NTG-tolerant rat aortas. Increased PDE1A1 was due to mRNA upregulation as measured by relative quantitative reverse transcription-polymerase chain reaction. The PDE1-specific inhibitor vinpocetine partially restored the sensitivity of the tolerant vasculature to subsequent NTG exposure. In cultured rat aortic VSMCs, angiotensin II (Ang II) increased PDE1A1 activity, and vinpocetine blocked the effect of Ang II on decrease in cGMP accumulation. CONCLUSIONS: Induction of PDE1A1 in nitrate-tolerant vessels may be one mechanism by which NO/cGMP-mediated vasodilation is desensitized and Ca(2+)-mediated vasoconstriction is supersensitized. Inhibiting PDE1A1 expression and/or activity could be a novel therapeutic approach to limit nitrate tolerance.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3.

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis.

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.

Downregulation of fasting-induced cAMP response element-mediated gene induction by leptin in neuropeptide Y neurons of the arcuate nucleus.

States of increased metabolic demand such as fasting modulate hypothalamic neuropeptide gene expression and decrease circulating leptin levels. This study tested the hypotheses that fasting stimulates gene induction mediated by cAMP response element (CRE)-dependent increases in gene transcription and that fasting-induced decreases in leptin can regulate this CRE-mediated gene induction. Using C57BL/6J mice transgenic for a CRE-lacZ construct, an immunocytochemical study showed that fasting activated reporter gene expression in the hypothalamic arcuate nucleus (Arc) in a small subset of neurons and increased phosphorylation of CRE binding protein. The increase of beta-galactosidase expression caused by fasting was inhibited by a protein kinase A inhibitor, Rp-8-Br-cAMPS, when the compound was microinjected into the medial basal hypothalamus, and enhanced by intraperitoneal injection of selective phosphodiesterase inhibitors. In situ hybridization studies showed that neuropeptide Y (NPY) mRNA levels increased in the Arc during fasting, whereas proopiomelanocortin (POMC) mRNA levels decreased. Double labeling of mRNA and beta-galactosidase immunoreactivity in the fasted brain indicated that the subpopulation of the neurons expressing beta-galactosidase all produced NPY but not POMC. To study the possible involvement of decreased circulating leptin during starvation on CRE-mediated gene induction, leptin was administered intraperitoneally to fasted mice. Leptin significantly attenuated both beta-galactosidase expression and NPY gene expression stimulated by fasting, suggesting that leptin inhibits fasting-stimulated NPY gene expression at least in part through downregulation of CRE-mediated gene induction in the Arc. Leptin-induced modification of CRE-mediated gene induction in the Arc may play an essential role in the central regulation of feeding behavior and energy expenditure.

The delta subunit of type 6 phosphodiesterase reduces light-induced cGMP hydrolysis in rod outer segments.

The delta subunit of the rod photoreceptor PDE has previously been shown to copurify with the soluble form of the enzyme and to solubilize the membrane-bound form (). To determine the physiological effect of the delta subunit on the light response of bovine rod outer segments, we measured the real time accumulation of the products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of delta subunit GST fusion protein (delta-GST) to this preparation caused a reduction in the maximal rate of cGMP hydrolysis in response to light. The maximal reduction of the light response was about 80%, and the half-maximal effect occurred at 385 nm delta subunit. Several experiments suggest that this effect was not due to the effects of delta-GST on transducin or rhodopsin kinase. Immunoblots demonstrated that exogenous delta-GST solubilized the majority of the PDE in ROS but did not affect the solubility of transducin. Therefore, changes in the solubility of transducin cannot account for the effects of delta-GST in the pH assay. The reduction in cGMP hydrolysis was independent of ATP, which indicates that it was not due to effects of delta-GST on rhodopsin kinase. In addition to the effect on cGMP hydrolysis, the delta-GST fusion protein slowed the turn-off of the system. This is probably due, at least in part, to an observed reduction in the GTPase rate of transducin in the presence of delta-GST. These results demonstrate that delta-GST can modify the activity of the phototransduction cascade in preparations of broken rod outer segments, probably due to a functional uncoupling of the transducin to PDE step of the signal transduction cascade and suggest that the delta subunit may play a similar role in the intact outer segment.

Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits.

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.

Cloning and characterization of two splice variants of human phosphodiesterase 11A.

Phosphodiesterase 11A (PDE11A) is a recently identified family of cAMP and cGMP hydrolyzing enzymes. Thus far, a single splice variant designated as PDE11A1 has been reported. In this study, we identify and characterize two additional splice variants of PDE11A, PDE11A2 and PDE11A3. The full-length cDNAs are 2,141 bp for PDE11A2 and 2205 bp for PDE11A3. The ORF of PDE11A2 predicts a protein of 576 aa with a molecular mass of 65.8 kDa. The ORF of PDE11A3 predicts a protein of 684 aa with a molecular mass of 78.1 kDa. Comparison of the PDE11A2 sequence with that of PDE11A1 indicates an additional 86 aa at the N terminus of PDE11A2. Part of this sequence extends the potential cGMP binding region (GAF domain) present in PDE11A1. Compared with PDE11A2, PDE11A3 has an additional 108 N-terminal amino acids. Sequence analysis of PDE11A3 indicates the presence of another GAF domain in this region. This diversification of regulatory sequences in the N-terminal region of PDE11A splice variants suggests the interesting possibility of differential regulation of these enzymes. Recombinant PDE11A2 and -A3 proteins expressed in the Baculovirus expression system have the ability to hydrolyze both cAMP and cGMP. The K(m) values for cAMP hydrolysis are 3.3 microM and 5.7 microM for PDE11A2 and PDE11A3, respectively. The K(m) values for cGMP hydrolysis are 3.7 microM and 4.2 microM for PDE11A2 and PDE11A3, respectively. Both PDEs showed a V(max) ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an IC(50) of 1.8 microM, and to zaprinast, with an IC(50) of 28 microM. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with IC(50) values of 0.82 and 5 microM for dipyridamole and zaprinast, respectively.

Infection of CD4+ memory T cells by HIV-1 requires expression of phosphodiesterase 4.

Using PCR to monitor HIV-1 RNA genome reverse transcription and nuclear import of preintegration complexes, we found that memory, but not naive, CD4+ T cells could support transport of HIV-1 DNA to nuclei upon TCR/CD3 and IL-2 stimulation. Moreover, memory CD4+ T cells, unlike naive CD4+ T cells, express high levels of phosphodiesterase 4 (PDE4) constitutively. Selective blocking of PDE4 activity inhibited IL-2R expression and thereby led to abolishing HIV-1 DNA nuclear import in memory T cells; however, full-length viral DNA synthesis was not affected. Thus, blocking PDE4 prevents initiation of HIV-1 DNA circle formation in T cells. The fact that PDE4 is expressed constitutively at higher levels in memory vs naive CD4+ T cells may help HIV-1 readily infect memory T cells.

Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions.

The past eighteen months have provided much progress in the cyclic nucleotide phosphodiesterase (PDE) field. Six new phosphodiesterase genes have been discovered and characterized. In addition, several new highly specific PDE inhibitors have been developed and approved for clinical use. Finally, new strategies have been employed to determine PDE function in model systems including the use of antisense oligonucleotide and disruption techniques.

Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A.

We report here the cloning, expression, and characterization of human PDE11A1, a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits

Cloning and characterization of PDE7B, a cAMP-specific phosphodiesterase.

A member of the phosphodiesterase (PDE)7 family with high affinity and specificity for cAMP has been identified. Based on sequence homologies, we designate this PDE as PDE7B. The full-length cDNA of PDE7B is 2399 bp, and its ORF sequence predicts a protein of 446 amino acids with a molecular mass of 50.1 kDa. Comparison of the predicted protein sequences of PDE7A and PDE7B reveals an identity of 70% in the catalytic domain. Northern blotting indicates that the mRNA of PDE7B is 5.6 kb. It is most highly expressed in pancreas followed by brain, heart, thyroid, skeletal muscle, eye, ovary, submaxillary gland, epididymus, and liver. Recombinant PDE7B protein expressed in a Baculovirus expression system is specific for cAMP with a K(m) of 0.03 microM. Within a series of common PDE inhibitors, it is most potently inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of 2.1 microM. It is also inhibited by papaverine, dipyridamole, and SCH51866 at higher doses. PDE7A and PDE7B exhibit the same general pattern of inhibitor specificity among the several drugs tested. However, differences in IC(50) for some of the drugs suggest that isozyme selective inhibitors can be developed.

Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes.

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.

cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its gamma and delta subunits.

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, alpha beta gamma2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, alpha beta gamma2 delta) can release and bind cGMP at both noncatalytic sites; the delta subunit is likely responsible for this difference in cGMP exchange rates. Removal of the delta and/or gamma subunits yields a catalytic alphabeta dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (KD >1 microM); cGMP binding affinity at the second site (KD approximately 60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of gamma subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the gamma subunit with the alphabeta dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.

Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDE10A.

We report here the cloning, expression, and characterization of a dual-substrate, cAMP and cGMP, cyclic nucleotide phosphodiesterase (PDE) from mouse. This PDE contains the consensus sequence for a PDE catalytic domain, but shares <50% sequence identity with the catalytic domains of all other known PDEs and, therefore, represents a new PDE gene family, designated PDE10A. The cDNA for PDE10A is 3, 370 nt in length. It includes a full ORF, contains three in-frame stop codons upstream of the first methionine, and is predicted to encode a 779-aa enzyme. At the N terminus PDE10A has two GAF domains homologous to many signaling molecules, including PDE2, PDE5, and PDE6, which likely constitute a low-affinity binding site for cGMP. PDE10A hydrolyzes cAMP with a Km of 0.05 microM and cGMP with a Km of 3 microM. Although PDE10A has a lower Km for cAMP, the Vmax ratio (cGMP/cAMP) is 4.7. RNA distribution studies indicate that PDE10A is expressed at highest levels in testis and brain.

CD3- and CD28-dependent induction of PDE7 required for T cell activation.

Costimulation of both the CD3 and CD28 receptors is essential for T cell activation. Induction of adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase-7 (PDE7) was found to be a consequence of such costimulation. Increased PDE7 in T cells correlated with decreased cAMP, increased interleukin-2 expression, and increased proliferation. Selectively reducing PDE7 expression with a PDE7 antisense oligonucleotide inhibited T cell proliferation; inhibition was reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase (PKA). Thus, PDE7 induction and consequent suppression of PKA activity is required for T cell activation, and inhibition of PDE7 could be an approach to treating T cell-dependent disorders.