Phosphodiesterase Enzyme Research Articles

Role of Phosphodiesterase Enzyme Modulation in Protection of Hepatotoxicity Induced by D-Galactosamine in Rats

Role of Phosphodiesterase Enzyme Modulation in Protection of Hepatotoxicity Induced by D-Galactosamine in Rats

Phosphodiesterase 5 inhibitors: are they cardioprotective?

A growing body of animal studies provides evidence for potential cardioprotective effects of inhibitors of the enzyme phosphodiesterase isoform 5. Infarct size reduction by administration of phosphodiesterase 5 inhibitors was described in various experimental models of ischaemia and reperfusion. Furthermore, potential beneficial effects were demonstrated in experimental models of congestive heart failure and left ventricular hypertrophy. Some of the observed effects resemble the basic mechanisms of ischaemic pre-conditioning, mimicking both acute and delayed effects. Other effects may be due to action on systemic and cardiac haemodynamics. Mechanisms and signalling pathways, characterized in some of the experimental models, appear to be complex: for instance, the rate of cyclic guanosine monophosphate (cGMP) synthesis and the functional compartmentalization of intracellular cGMP metabolism as well as interaction with ss-adrenergic and nitric oxide signalling may influence effects in different experimental settings. In this review, we discuss mechanisms, signalling pathways, and experimental limitations and touch on considerations for translation into potentially useful applications in the clinical arena.

Lodenafil Carbonate: Reproductive and Developmental Evaluation

Lodenafil, is one new orally‐active, potent and selective inhibitor of the enzyme phosphodiesterase 5 (PDE5) developed in Brazil, with therapeutically application in erectile dysfunction. The objective of this study was to evaluate the reproduction toxicity and in offspring, evidencing specifically the occurrence of possible toxic effects in the fertility, gestation, maternal behavior and lactation, as well as growth and postnatal development of F1 since its birth, up to 4 days. Wistar rats received daily: metilcelulose, (2 control groups); 5,7mg/kg, 28,4mg/kg and 142mg/kg (2 groups of 10 males and 10 females in each dose).Drug administration occurred during 35 and 14 consecutive days for males and females, respectively. Animals had been observed for detention of possible clinical and behavior alterations, delivery, and other toxic signals including morbidity or mortality, gestation time, litter size number of each sex, total living pups and corporal weight of the offspring.Litter components had been analyzed in the maximum 24 h after the birth, and the evaluation repeated in all first 4 days after‐birth. No remarkable toxicity was found in the offspring even in the presence of maternal toxicity, no significant decrease in body weight gain was observed, decrease in offspring righting reflex and increase in litter size, were observed.FellowshipCristália Produtos Químicos e Farmacêuticos, Instituto Royal

Long-acting oral phosphodiesterase inhibition preconditions against reperfusion injury in an experimental lung transplantation model

Ischemia-reperfusion injury remains a devastating complication of lung transplantation. Phosphodiesterase inhibitors have been shown to precondition tissues against ischemia-reperfusion injury. Little is known, however, about the utility of phosphodiesterase inhibition in reperfusion injury after lung transplantation. We evaluated the long-acting phosphodiesterase-5 inhibitor, tadalafil, in an ex vivo lung transplant model. New Zealand White rabbits (4 kg), were given oral tadalafil (n = 11) 24 hours before lung harvest and compared with rabbits given oral vehicle alone (n = 11). Lungs were recovered with Perfadex solution (Vitrolife, Kungsbacka, Sweden) and cold stored for 18 hours. After storage, lung blocks were reperfused with donor rabbit blood in an ex vivo apparatus. Pulmonary artery pressures were recorded with serial arterial and venous blood gas sampling and animals served as their own controls. Phosphodiesterase-5 and protein kinase G tissue activity assays confirmed drug effects. Luminol chemiluminescence assay was used to measure reactive oxygen species and levels of endothelial and inducible nitric oxide synthase were measured. Extended cold storage, followed by reperfusion produced a consistent reproducible decrease in oxygenation and increase in pulmonary pressure. Tadalafil-treated animals exhibited greater Pao(2) throughout the course of reperfusion (P = .001) Mean pulmonary artery pressure was lower in tadalafil-treated animals (22 vs 40 mm Hg; P = .04). Phosphodiesterase-5 activity was decreased (143 +/- 8 vs 205 +/- 32 mP; P < .001) with protein kinase G activity increased (25 +/- 12 vs 12 +/- 2.4 fU/microg; P = .01) in the experimental group confirming that oral pretreatment resulted in active phosphodiesterase inhibition in the lung tissue. Reactive oxygen species (as measured by luminol activity) were decreased in tadalafil-treated animals (7.8 +/- 1.5 vs 10.2 +/- 1.2 relative light units; P = .003). Our experimental model demonstrates that oral donor pretreatment with a long-acting phosphodiesterase inhibitor is an effective strategy for improving pulmonary performance after reperfusion. Importantly, phosphodiesterase enzymes and their downstream effectors may play a critical role in reperfusion injury after lung transplantation.

Diffusion in narrow domains and application to phototransduction

The mean time for a Brownian particle to find a small target inside a narrow domain is a key parameter for many chemical reactions occurring in cellular microstructures. Although current estimations are given for a large class of domains, they cannot be used for narrow domains often encountered in cellular biology, such as the synaptic cleft, narrow compartments in the outer segment of vertebrate photoreceptors, or neuron-glia contact. We compute here the mean time for a Brownian particle to hit a small target placed on the surface of a narrow cylinder. We then use this result to estimate the rate constant of cyclic-GMP (cGMP) hydrolysis by the activated enzyme phosphodiesterase (PDE) in the narrow microdomains that build up the outer segment of a rod photoreceptor. By controlling the cGMP concentration, PDE activity is at the basis of the early photoresponse chemical reaction cascade. Our approach allows us to compute the cGMP rate constant as a function of biophysical parameters.

Caboxamycin, a new antibiotic of the benzoxazole family produced by the deep-sea strain Streptomyces sp. NTK 937

Caboxamycin, a new benzoxazole antibiotic, was detected by HPLC-diode array screening in extracts of the marine strain Streptomyces sp. NTK 937, which was isolated from deep-sea sediment collected in the Canary Basin. The structure of caboxamycin was determined by mass spectrometry, NMR experiments and X-ray analysis. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and the enzyme phosphodiesterase.

Heart Failure in Children

Why is heart failure in children important? If we just consider the number of individuals affected, adult heart failure is clearly a more compelling public health problem. However, the relatively small numbers belie the overall economic and social impact of pediatric heart failure. When a child is admitted to the hospital for heart failure, the costs are considerably higher for children than adults because of the frequent need for surgical or catheter-based intervention. The demands of medical care can fray the family structure and adversely affect parental economic productivity. When a child dies of heart failure, the economic impact is magnified enormously because of the number of potentially productive years lost per death. For these and other reasons, heart failure in children is a serious public health concern. In addition, growing numbers of children with heart failure are reaching adulthood because of the successful application of medical and surgical heart failure therapies and the improved outcomes of congenital heart surgery. A greater understanding of the pathophysiology of heart failure in childhood may help inform therapeutic strategies once these children become adults. Furthermore, given the recent explosion of research in the impact of cardiac development and cardiogenetics on pediatric cardiovascular disease, it is not outside the realm of possibility for a pediatric discovery to be made that will also benefit adults with heart failure. We may not yet be able to agree on a definition of heart failure, but the cardinal symptoms, dyspnea, anasarca (“dropsy”), and cachexia, were well recognized in antiquity.1 These symptoms were not specific for cardiac pathology, and it was not until the 17th century, when William Harvey definitively identified the heart as an organ that pumped blood rather than generating heat, that the heart could begin to be understood as a source of dyspnea, edema, and …

Water Treatment Residuals and Biosolids Co‐applications Affect Phosphatases in a Semi‐arid Rangeland Soil

Co‐application of biosolids and water treatment residuals (WTR) land has not been extensively studied but may be beneficial by sorbing excess biosolid‐borne or soil phosphorus (P) onto WTR, reducing the likelihood of off‐site movement. Reduction of excess soil P may affect the role of specific P‐cleaving enzymes. The research objective was to understand the long‐term effects of single co‐applications and the short‐term impacts of repeated co‐applications on soil acid phosphomonoesterase, phosphodiesterase, pyrophosphatase, and phytase enzyme activities. Test plots were 7.5 × 15 m with treatments consisting of three different WTR rates with a single biosolids rate (5, 10, and 21 Mg WTR ha−1; 10 Mg biosolids ha−1) surface co‐applied once in 1991 or reapplied in 2002. Control plots consisted of those that received no WTR–biosolids co‐applications and plots that received only 10 Mg biosolids ha−1. Plots were sampled to a 5‐cm depth in 2003 and 2004, and soil phosphatases and phytase enzyme activities were measured. Soil phosphodiesterase activity decreased in WTR‐amended plots, and pyrophosphatase activity decreased with increasing WTR application rates. In contrast, acid phosphatase and phytase activity increased with WTR addition, with WTR application possibly triggering a deficiency response causing microorganisms or plants to secrete these enzymes. Biosolids and WTR co‐applications may affect enzymatic strategies for P mineralization in this study site. Reductions in phosphodiesterase activity suggest less P mineralization from biomass sources, including nucleic acids and phospholipids. Increased acid phosphatase and phytase activities indicate that ester‐P and inositol‐P may be important plant‐available P sources in soils amended with WTR.

Clinical Use of Phosphodiesterase-5 Inhibitors in Chronic Heart Failure

Received June 25, 2008; accepted September 16, 2008. Nowadays, the inhibitors of the phosphodiesterase enzyme type 5 (PDE5), thanks to a sustained relaxant activity on smooth muscles of the corpus cavernosum, are a widely used remedy for erectile dysfunction in man. This class of compounds was originally developed for the treatment of angina pectoris, based on the ability of oversignaling the pathway originated from nitric oxide (NO) and pursued via cGMP signaling. Because the clinical effects were not as promising as initially reported by experimental studies, PDE5 inhibitors1 were not regarded as a remarkable advancement over commonly used nitrates. Subsequently, however, it was realized that the clinical applicability of the enhanced NO/cGMP pathway by inhibiting the PDE5 activity could be larger than previously thought. Over the last few years, the use of PDE5 inhibitors has been expanded to the therapeutic management of other cardiovascular disorders, including chronic heart failure (CHF). Their clinical applicability to CHF is the subject of this review. The homeostatic role of phosphodiesterases (PDEs) as related to the intracellular levels of cAMP and cGMP was first described by Sutherland,2 who, because of this, was awarded the Nobel Prize for Physiology and Medicine in 1971. These enzymes hydrolyze the phosphodiester bond of cAMP and cGMP to form the inactive 5′-AMP and 5′-GMP. In optimizing the intracellular levels of cAMP and cGMP, breakdown is predominant over synthesis. PDEs comprise a superfamily with 11 subfamilies, which have been characterized on the basis of amino acid sequence, substrate specificity, pharmacological properties and allosteric regulation. Within these families, more than 40 isoforms are expressed either by different genes or as expression of the same gene through alternative splicing.3 The substrate specificities include the enzymes that are specific for cAMP hydrolysis, those for cyclic GMP hydrolysis,4 and those that …

Postulated vasoactive neuropeptide immunopathology affecting the blood&amp;ndash;brain/blood&amp;ndash;spinal barrier in certain neuropsychiatric fatigue-related conditions: A role for phosphodiesterase inhibitors in treatment?

Neuropsychiatric symptoms occur in a number of neurological fatigue-related conditions including multiple sclerosis (MS), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and chronic fatigue syndrome (CFS). These conditions have been attributed variably to neuroinflammatory and neurodegenerative processes. While autoimmune pathology, at least in part, has long been suspected in these conditions proof has been elusive. Autoimmune pathomechanisms affecting the blood-brain barrier (BBB) or blood-spinal barrier (BSB) may predispose the BBB/BSB to 'leakiness' and be a precursor to additional autoimmune events resulting in neuroinflammatory or neurodegenerative processes. The aim of the paper is to postulate immunopathology of the cerebrospinal perivascular compartment involving certain vasoactive neuropeptides, specifically pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), in the etiology of certain neuropsychiatric fatigue-related conditions such as MS, ALS, PD, and CFS. Vasoactive neuropeptides (VNs) such as PACAP and VIP have critical roles as neurotransmitters, vasodilators including perfusion and hypoxia regulators, and immune and nociception modulators. PACAP and VIP are widely distributed in the central nervous system (CNS) and have key roles in CNS blood vessels including maintaining functional integrity of the BBB and BSB. Autoimmunity affecting these VNs would likely have a detrimental effect on BBB and BSB functioning arguably predisposing to further pathological processes. Virchow-Robin spaces (VRS) are perivascular compartments surrounding small vessels within the CNS which contribute to the BBB and BSB integrity and contain PACAP and VIP receptors. Autoimmunity of these receptors would likely affect BBB and VRS function and therefore may contribute to the etiology of these conditions by affecting CNS and immunological homeostasis, including promoting neuropsychological symptomatology. PACAP and VIP, as potent activators of adenylate cyclase (AC), have a key role in cyclic adenosine monophosphate (cAMP) production affecting regulatory T cell (Treg) and other immune functions. Phosphodiesterase enzymes (PDEs) catalyze cAMP and PDE inhibitors (PDEIs) maintain cAMP levels and have proven and well known therapeutic benefit in animal models such as experimental allergic encephalomyelitis (EAE). Therefore PDEIs may have a role in therapy for certain neuropsychiatric fatigue-related conditions.

PDE type-4 inhibition increases L-type Ca2+ currents, action potential firing, and quantal size of exocytosis in mouse chromaffin cells

We studied the effects of the cAMP-hydrolyzing enzyme phosphodiesterase type-4 (PDE4) on the L-type Ca(2+) channels (LTCCs) and Ca(2+)-dependent secretion in mouse chromaffin cells (MCCs). The selective PDE4 inhibitor rolipram (3 microM) had a specific potentiating action on Ca(2+) currents of MCCs (40% increase within 3 min). A similar effect was produced by the selective beta(1)-AR agonist denopamine (1 microM) and by the unselective PDEs inhibitor IBMX (100 microM). Rolipram and denopamine actions were selective for LTCCs, and the Ca(2+) current increase remained unchanged if the two compounds were applied simultaneously. This suggests that at rest, LTCCs in MCCs are down-regulated by the low levels of cAMP determined by PDE4 activity and that LTCCs can be up-regulated by either inhibiting PDE4 or activating beta(1)-AR. No other PDEs are likely involved in this specific action. PDE4 inhibition had also a marked effect on the spontaneous firing of resting MCCs and catecholamine secretion. Rolipram up-regulated the LTCCs contributing to the "pace-maker" current underlying action potential (AP) discharges and accelerated the firing rate, with no significant effects on AP waveform. Acceleration of AP firing was also induced by the LTCC-agonist Bay K (1 microM), while nifedipine (3 microM) reduced the firing frequency, suggesting that LTCCs and intracellular cAMP play a key role in setting the pace-maker current regulating MCCs excitability. Rolipram increased also the size of the ready-releasable pool and the quantal content of secretory vesicles without affecting their probability of release. Thus, rolipram acts on MCCs by up-regulating both exocytosis and AP firings. These two processes are effectively down-regulated by PDE4 at rest and can dramatically increase the quantity of released catecholamines when PDE4 is inhibited and/or cAMP is raised.

A Cell-Based PDE4 Assay in 1536-Well Plate Format for High-Throughput Screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.

Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis.

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum. Here we investigate the role of specific GC and PDE isoforms in gamete formation – a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum. Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation – the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCβ gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEδ gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEδ plays a crucial role in regulating cGMP levels during sexual development.

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a recently discovered enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo following the DNA processing activity of topoisomerase I (Top1). For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes, which can be generated by either exogenous or endogenous factors. Tdp1 has been regarded as a potential therapeutic co-target of Top1 in that it seemingly counteracts the effects of Top1 inhibitors, such as camptothecin and its clinically used derivatives. Thus, by reducing the repair of Top1-DNA lesions, Tdp1 inhibitors have the potential to augment the anticancer activity of Top1 inhibitors provided there is a presence of genetic abnormalities related to DNA checkpoint and repair pathways. Human Tdp1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolates and 3'-abasic sites indicating it may function as a general 3'-DNA phosphodiesterase and repair enzyme. The importance of Tdp1 in humans is highlighted by the observation that a recessive mutation in the human TDP1 gene is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1). This review provides a summary of the biochemical and cellular processes performed by Tdp1 as well as the rationale behind the development of Tdp1 inhibitors for anticancer therapy.

Use of a column-switching high-performance liquid chromatography method to assess the presence of specific binding of (R)- and (S)-[11C]rolipram and their labeled metabolites to the phosphodiesterase-4 enzyme in rat plasma and tissues

To complement recent studies using the high-affinity (11)C-labeled phosphodiesterase-4 (PDE4) inhibitor (R)-rolipram and the less active enantiomer (S)-[(11)C]rolipram for in vivo quantification of PDE4 levels, we evaluated the presence of radiolabeled metabolites and their potential binding to PDE4 in the rat plasma, brain, heart, pancreas, skeletal muscle and brown adipose tissue. A reverse-phase capture and analytical HPLC column-switch method was used to detect (R)-[(11)C]rolipram, (S)-[(11)C]rolipram and their radiolabeled metabolites in rat plasma and tissue extracts. The relative proportion of PDE4-specific binding of the radiotracers and their labeled metabolites was analyzed following co-injections with a saturating dose of unlabeled (R)-rolipram at 45 min post-tracer injection in tissue extracts. Radiolabeled metabolites were found in the plasma (72-75% of total radioactive signal), and in the heart, skeletal muscle, pancreas and brown adipose tissue (44-52%), but not in the brain. In comparison to polar labeled metabolites, the proportion of unchanged (R)-[(11)C]rolipram was reduced in PDE4-rich organs by co-injection of unlabeled (R)-rolipram. Conversely, no changes were obtained in brown adipose tissue, or with (S)-[(11)C]rolipram, suggesting that radiolabeled metabolites of (R)-[(11)C]rolipram display no specific binding to PDE4. Radiolabeled hydrophilic metabolites are unlikely to compete with (R)-[(11)C]rolipram for PDE4-specific retention. However, due to the high proportion of the radioactive metabolites in the total radioactive signal, any kinetic modeling calculations in the peripheral tissues will need to take into account the presence of labeled metabolites.

Murine and Human Autotaxin α, β, and γ Isoforms: GENE ORGANIZATION, TISSUE DISTRIBUTION, AND BIOCHEMICAL CHARACTERIZATION

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.

Feasibility of using a surface plasmon resonance-based biosensor to detect and quantify yessotoxin

Yessotoxin (YTX) is a disulfated polyether toxin produced by marine dinoflagellates. Although there is no clear evidence that YTX is toxic to humans, it is a major cause of false positives in DSP toxin detection by mouse bioassay. We developed a new detection and quantification method for yessotoxin using a BiaCore X Surface plasmon resonance (SPR)-based biosensor. The assay is based in the interaction of YTX with phosphodiesterase enzymes (PDE), one of its cellular targets. The injection of several YTX concentrations (3–12 μM) over immobilized PDE I, showed a dose dependent binding signal, which K obs (observed rate constant) allowed us to obtain a calibration curve with a linear fit. The detection of yessotoxin using SPR-based biosensor allows the quantification of the toxin with an automated and repetitive method at concentrations in the range of the 1 mg kg −1 European regulatory limit.

Molecular pharmacology of adipocyte-secreted autotaxin

Autotaxin is a type II ecto-nucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that autotaxin also catalyses a lyso-phospholipase D activity. This enzyme probably provides most of the extracellular lyso-phosphatidic acid from lyso-phosphatidylcholine. There is almost no pharmacological tools available to study autotaxin. Indeed, all the reported inhibitors, thus far, are uneasy-to-use, lyso-phosphatidic acid derivatives. Initially, autotaxin was recognized as a phosphodiesterase (NPP2) [Bollen et al., Curr. Rev. Biochem. Biol. 35 (2000) 393–432], based on sequence similarity and enzymatic capability of autotaxin to catalyse ecto-nucleotidase activity. Phosphodiesterase forms a large family of enzymes characterized by a large number of chemically diverse inhibitors. None of them have been tested on autotaxin activity. For this reason, we screened those reported inhibitors, as well as a series of compounds, mostly kinase inhibitor-oriented, on autotaxin activity. Only two compounds of the various phosphodiesterase inhibitors (calmidazolium and vinpocetine) were potent enough to inhibit autotaxin catalytic activity. From the kinase inhibitor library, we found damnacanthal and hypericin, inhibiting phosphodiesterase activity in the 100-μM range, comparable to most of other available phospholipid-like inhibitors.

Increased Expression of the cGMP-Specific Phophodiesterase, PDE9A, in Sickle Cell Disease (SCD) Reticulocytes and Neutrophils, and Induction of Erythroid γ Globin Expression and Reduction of SCD Neutrophil Adhesion Following PDE9A Inhibition.

Modulation of the levels of the NO second messenger, cyclic guanosine monophosphate (cGMP), has been identified as a possible therapeutic approach for SCD treatment. Up-regulation of cGMP-dependent signaling may induce γ-globin production in erythrocytic lineage cells and diminish the adhesive properties of leukocytes, the adhesion of which contributes to vaso-occlusion. Since the phosphodiesterase (PDE) enzymes, which degrade intracellular cyclic nucleotides, display differing cellular expressions, their inhibition can provide tissue-specific induction of cGMP. We studied the gene expressions of 3 types of cGMP-specific PDE (−1A, −5A, −9A) in the reticulocytes (ret) and neutrophils (neu) of healthy controls (AA), steady-state SCD patients (SS) and SCD patients on hydroxyurea therapy (SSHU; 20–30 mg/kg/day). Ret and neu were separated from whole blood before extracting mRNA, synthesizing cDNA and quantifying PDE gene expression by Real-Time PCR relative to β-actin and GAPDH expression. PDE1 was not found expressed in ret of any of the groups, but was found expressed in the neu of all three groups (≤14.2 rel expression). Intermediate PDE5 expression was observed in ret of all groups (≤16.8 rel express); PDE5 expression in neu was, however, almost undetectable in all groups (<0.3 rel. express). In contrast, high PDE9A expressions were found in ret of all groups with significantly higher expression (P<0.05, unpaired t test) in the ret of SS than SSHU and AA (68.3±10.4; 181.8±50.0; 54.8±22.2 rel express for AA, SS, SSHU, resp., n=6). Extremely high PDE9A expressions were also found in the neu of all groups, although PDE9A was significantly higher in SCD neu than in control neu (101.7±24.0; 722.8±102.2; 873.9±221.7 rel. express for AA, SS, SSHU, resp., n≥4, P<0.05 for SS/SSHU comp. AA). Comparison of PDE9A expression in diverse cell types that included T98G glial cells, liver, colon, skin, spleen, lymphnode, mammary, ovary, uterus, testicle and K562 erythroleukemic cells demonstrated very low level or virtually undetectable gene PDE9A expression in all of these tissue types (<6.7 rel express in all tissues), with the exception of K562 cells (96.8 rel. express). Data indicate that PDE9A may be expressed at higher levels in hematopoietic lineage cells. In keeping with our results, co-culture of K562 cells in the presence of the PDE1 and PDE5 inhibitors, vinpocetine (1–50μM) and sildenafil (50–200μg/ml), respectively, did not significantly increase intracellular cGMP, nor γ-globin expression (data not shown, P>0.05), suggesting that PDE1 and PDE5 are not suitable targets for pharmacological inhibition in erythroid lineage cells. In contrast, co-culture of K562 cells in the presence of the PDE9A-specific inhibitor, BAY 73-6691, for 48 h significantly increased intracellular γ-globin expression by 97.0±7.8% (1.0μM, n=3, P<0.05). Furthermore, in vitro static adhesion assays showed that co-incubation of SS neu with BAY 73-6691 reversed augmented SS neu adhesion to immobilized fibronectin (20μg/ml) to control neu adhesion levels (AA neu adhesion: 8.7±1.3%; SS neu adhesion: 12.6±1.4%, reduced to 7.2±1.3% with 1.0 μM BAY 73-6691, n=4, P<0.01 comp.basal SS adhesion). The PDE9A enzyme may constitute a relatively hematopoietic cell-specific target for pharmacological inhibition with a potential to induce γ-globin production and to inhibit cell adhesion mechanisms, with beneficial consequences for individuals with SCD.

CAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling

The PDE4 (phosphodiesterase-4) enzyme family consists of a distinct array of N-terminal splice variant isoforms arising from four subfamily genes (4A, 4B, 4C and 4D). These all hydrolyse specifically the intracellular second messenger cAMP. Although identical in catalytic function, each isoform appears to serve a non-superfluous regulatory role. For example, a beta-arrestin-sequestered subpopulation of the PDE4D5 isoform specifically regulates the phosphorylation of the beta(2)-AR (beta(2)-adrenergic receptor) by PKA (protein kinase A; also called cAMP-dependent protein kinase). This was elucidated by the use of novel technologies, including dominant-negative approaches, siRNA (small interfering RNA) knockdown and spot-immobilized peptide array analyses. Functional phenotypes uncovered using these methodologies have shown that beta-arrestin-sequestered PDE4D5 shapes the spatial cAMP gradient around the membrane-bound beta(2)-AR, regulating its phosphorylation by PKA and its ability to activate ERK (extracellular-signal-regulated kinase) through G(i) in cardiomyocytes and HEK-293 (human embryonic kidney)-B2 cells. This approach has provided the very first identification of a non-redundant and specific role for a PDE isoform. The fact that phenotypes can be uncovered by displacing PDE4 isoforms from specific anchor sites using dominant-negative constructs and cell-permeable peptides points to novel means for developing therapeutics aimed at disrupting specifically sequestered PDE isoforms and even specifically sequestered subpopulations of individual isoforms.