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Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH., (© 2023 Miyagawa et al.)

2'-Amino-locked nucleic acid has a functionalizable nitrogen atom at the 2'-position of its furanose ring that can provide desired properties to a nucleic acid as a scaffold. In this study, we synthesized a novel nucleic acid, 2'-N-methanesulfonyl-2'-amino-locked nucleic acid (ALNA[Ms]) and conducted comparative studies on the physical and pharmacological properties of the ALNA[Ms] and on conventional nucleic acids, such as 2'-methylamino-LNA (ALNA[Me]), which is a classical 2'-amino-LNA derivative, and also on 2',4'-BNA/LNA (LNA). ALNA[Ms] oligomers exhibited binding affinities for the complementary RNA strand that are similar to those of conventional nucleic acids. Four types of ALNA[Ms] nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. The knockdown abilities of Malat1 RNA using the Matat1 antisense oligonucleotide (ASO) containing ALNA[Ms] were higher than those of ALNA[Me] and were closer to those of LNA. Furthermore, the ASO containing ALNA[Ms] showed different tissue tropism from that containing LNA. ALNA[Ms] exhibited biological activities that were distinct from conventional constrained nucleic acids, suggesting the possibility that ALNA[Ms] can serve as novel modified nucleic acids in oligonucleotide therapeutics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33-deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR-33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR-33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride (CaCl 2 )-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA 1 week after CaCl 2 administration to examine the effect of anti-miR-33b. Histological examination revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved compared with mice treated with control oligonucleotides. These results support that inhibition of miR-33b is effective in the treatment for AAA., (© 2022. The Author(s).)

Guanidine-bridged nucleic acid (GuNA) is a novel 2',4'-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) analog containing cations that exhibit strong affinity for target RNA and superior nuclease resistance. In this study, Malat1 antisense oligonucleotide (ASO) bearing GuNA was evaluated for target knockdown (KD) activity and tolerability. The GuNA ASO did not interfere with RNase H recruitment on the target RNA/ASO heteroduplex and did show potent target KD activity in a skeletal muscle-derived cell line equivalent to that of the LNA ASO under gymnotic conditions, whereas almost no KD activity was observed in a hepatocyte-derived cell line. The GuNA ASO exhibited potent KD activity in various tissues; the KD activity in the skeletal muscle was equivalent with that of the LNA ASO, but the KD activities in the liver and kidney were clearly lower compared with the LNA ASO. In addition, despite the higher accumulation of the GuNA ASO in the liver, levels of aspartate aminotransferase and alanine aminotransferase with the GuNA ASO administration were not elevated compared with those induced by the LNA ASO. Our data indicate that the GuNA ASO is tolerable and exhibits unique altered pharmacological activities in comparison with the LNA ASO in terms of the relative effect between liver and skeletal muscle.

If an object is photographed at motion in front of a static background, the object will be blurred while the background sharp and partially occluded by the object. The goal is to recover the object appearance from such blurred image. We adopt the image formation model for fast moving objects and consider objects undergoing 2D translation and rotation. For this scenario we formulate the estimation of the object shape, appearance, and motion from a single image and known background as a constrained optimization problem with appropriate regularization terms. Both similarities and differences with blind deconvolution are discussed with the latter caused mainly by the coupling of the object appearance and shape in the acquisition model. Necessary conditions for solution uniqueness are derived and a numerical solution based on the alternating direction method of multipliers is presented. The proposed method is evaluated on a new dataset.

Phosphodiesterase 10A (PDE10A) is a dual-substrate PDE that hydrolyzes both cAMP and cGMP. PDE10A is selectively expressed in medium spiny neurons in the striatum, suggesting the potential of PDE10A inhibitors in the treatment of schizophrenia. This study presents the pharmacological profile of a novel PDE10A inhibitor, 2-[(E)-2-(7-fluoro-3-methylquinoxalin-2-yl)vinyl]-6-pyrrolidin-1-yl-N-(tetrahydro-2H-pyran-4-yl)pyrimidin-4-amine hydrochloride (T-251) in rodent models of schizophrenia. T-251 showed a potent inhibitory activity against human PDE10A (IC 50  = 0.050 nmol/L) and showed high selectivity over other PDE families which have over 10,000-fold IC 50 values. Oral administration of T-251 (0.1-1.0 mg/kg) increased cAMP and cGMP in the striatum in a dose-dependent manner. Oral administration of T-251 attenuated MK-801 induced hyperactivity (ED 50  = 0.68 mg/kg) and suppressed conditioned avoidance response (ID 50  = 0.87 mg/kg) in rats in a dose dependent manner. Furthermore, T-251 significantly attenuated MK-801 induced prepulse inhibition deficits and cognitive deficits in rats. Unlike haloperidol and olanzapine, T-251 (1.0-30 mg/kg) did not cause catalepsy in rats. Moreover, T-251 (0.6 and 6.0 mg/kg) did not increase plasma levels of prolactin at 1 h after administration, whereas haloperidol and olanzapine significantly increased them. The antipsychotic-like effects and cognitive enhancement of T-251 without catalepsy or plasma prolactin elevation observed in rats suggests that T-251 would be a novel antipsychotic with an improved side-effect profile., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We have developed a new class of PDE10A inhibitor, a pyrazolo[1,5-a]pyrimidine derivative MT-3014 (1). A previous compound introduced was deprioritized due to concerns for E/Z-isomerization and glutathione-adduct formation at the core stilbene structure. We discovered pyrazolo [1,5-a] pyrimidine as a new lead scaffold by structure-based drug design utilizing a co-crystal structure with PDE10A. The lead compound was optimized for in vitro activity, solubility, and selectivity against human ether-á-go-go related gene cardiac channel binding. We observed that MT-3014 shows excellent efficacy in rat conditioned avoidance response test and suitable pharmacokinetic properties in rats, especially high brain penetration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Phosphodiesterase (PDE) 10A is a dual hydrolase of cAMP and cGMP and highly expressed in striatal medium spiny neurons. Inhibition of PDE10A modulates the activity of medium spiny neurons (MSN) via the regulation of cAMP and cGMP. Signal control of MSN is considered associated with psychotic symptoms. Therefore PDE10A inhibitor is expected as a therapeutic method for psychosis disease such as schizophrenia. Avanafil (1) is a PDE5 inhibitor (treatment for erectile dysfunction) discovered by our company. We paid attention to the homology of PDE10A and PDE5 and took advantage of PDE5 inhibitor library to discover PDE10A inhibitors, and found a series of compounds that exhibit higher potency for PDE10A than PDE5. We transformed the afforded derivatives, which had weak inhibitory activity against PDE10A, and discovered stilbene as a PDE10A inhibitor. Brain penetration of this compound was improved by further conversion of N-containing heterocycles and their substituents. The afforded dimethylaminopyrimidine was effective for rat conditioned avoidance response (CAR) test; however, it did not exhibit good brain penetration. We performed in-depth optimization focusing on substituents of the quinoxaline ring, and produced 3-methyl-7-fluoro quinoxaline. This compound was the most effective in rat CAR test due to its strong PDE10A inhibitory activity and good pharmacokinetics.

Blind deconvolution is a strongly ill-posed problem comprising of simultaneous blur and image estimation. Recent advances in prior modeling and/or inference methodology led to methods that started to perform reasonably well in real cases. However, as we show here, they tend to fail if the convolution model is violated even in a small part of the image. Methods based on variational Bayesian inference play a prominent role. In this paper, we use this inference in combination with the same prior for noise, image, and blur that belongs to the family of independent non-identical Gaussian distributions, known as the automatic relevance determination prior. We identify several important properties of this prior useful in blind deconvolution, namely, enforcing non-negativity of the blur kernel, favoring sharp images over blurred ones, and most importantly, handling non-Gaussian noise, which, as we demonstrate, is common in real scenarios. The presented method handles discrepancies in the convolution model, and thus extends applicability of blind deconvolution to real scenarios, such as photos blurred by camera motion and incorrect focus.

This paper introduces a set of methods for image and video forensic analysis. They were designed to help to assess image and video credibility and origin and to restore and increase image quality by diminishing unwanted blur, noise, and other possible artifacts. The motivation came from the best practices used in the criminal investigation utilizing images and/or videos. The determination of the image source, the verification of the image content, and image restoration were identified as the most important issues of which automation can facilitate criminalists work. Novel theoretical results complemented with existing approaches (LCD re-capture detection and denoising) were implemented in the PIZZARO software tool, which consists of the image processing functionality as well as of reporting and archiving functions to ensure the repeatability of image analysis procedures and thus fulfills formal aspects of the image/video analysis work. Comparison of new proposed methods with the state of the art approaches is shown. Real use cases are presented, which illustrate the functionality of the developed methods and demonstrate their applicability in different situations. The use cases as well as the method design were solved in tight cooperation of scientists from the Institute of Criminalistics, National Drug Headquarters of the Criminal Police and Investigation Service of the Police of the Czech Republic, and image processing experts from the Czech Academy of Sciences., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Jasmonates are plant lipid-derived oxylipins that act as key signaling compounds in plant immunity, germination, and development. Although some physiological activities of natural jasmonates in mammalian cells have been investigated, their anti-inflammatory actions in mammalian cells remain unclear. Here, we investigated whether jasmonates protect mouse microglial MG5 cells against lipopolysaccharide (LPS)-induced inflammation. Among the jasmonates tested, only 12-oxo-phytodienoic acid (OPDA) suppressed LPS-induced expression of the typical inflammatory cytokines interleukin-6 and tumor necrosis factor α. In addition, only OPDA reduced LPS-induced nitric oxide production through a decrease in the level of inducible nitric oxide synthase. Further mechanistic studies showed that OPDA suppressed neuroinflammation by inhibiting nuclear factor κB and p38 mitogen-activated protein kinase signaling in LPS-activated MG5 cells. In addition, OPDA induced expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of inflammation, in MG5 cells. Finally, we found that the nuclear factor erythroid 2-related factor 2 signaling cascade induced by OPDA is not involved in the anti-inflammatory effects of OPDA. These results demonstrate that OPDA inhibited LPS-induced cell inflammation in mouse microglial cells via multiple pathways, including suppression of nuclear factor κB, inhibition of p38, and activation of SOCS-1 signaling., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

A novel series of highly selective phosphodiesterase 5 (PDE5) inhibitors was found. 8H-Pyrido[2,3-d]pyrimidin-7-one derivatives bearing an (S)-2-(hydroxymethyl)pyrrolidin-1-yl group at the 2-position and a 3-chloro-4-methoxybenzyl group at the 8-position exhibited potent PDE5 inhibitory activities and high PDE5 selectivity over PDE6. Among the synthesized compounds, the 5-methyl analogue (5b) showed the most potent relaxant effect on isolated rabbit corpus cavernosum with an EC30 value of 0.85 nM., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Jasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity., Methods: The cells were pretreated with individual jasmonates for 24h and exposed to hydrogen peroxide (H2O2) for 24h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA)., Results: Among the jasmonates, only OPDA suppressed H2O2-induced cytotoxicity. OPDA pretreatment also inhibited the H2O2-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2., Conclusions: These results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway., General Significance: Plant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Novel pyrimidine-5-carboxamide derivatives bearing a 3-chloro-4-methoxybenzylamino group at the 4-position were identified as potent and highly selective phosphodiesterase 5 inhibitors. Among them, we successfully found 10j (avanafil) which exhibited a potent relaxant effect on isolated rabbit cavernosum (EC30=2.1 nM) and a high isozyme selectivity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

5-(3,4,5-Trimethoxybenzoyl)-4-amimopyrimidine derivatives were found as a novel chemical class of potent and highly selective phosphodiesterase 5 inhibitors. A pseudo-ring formed by an intramolecular hydrogen bond constrained the conformation of 3-chloro-4-methoxybenzylamino and 3,4,5-trimethoxybenzoyl substituents and led to the discovery of T-6932 (19a) with a potent PDE5 inhibitory activity (IC50 = 0.13 nM) and a high selectivity over PDE6 (IC50 ratio: PDE6/PDE5 = 2400). Further modification at the 2-position of T-6932 resulted in the finding of 26, which exhibited potent relaxant effects on isolated rabbit corpus cavernosum (EC30 = 11 nM) with a high PDE5 selectivity over PDE6 (IC50 ratio: PDE6/PDE5 = 2800)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The myelin sheath consists of a unique multiple layer structure that acts as an insulator between neuronal axons to enhance the propagation of the action potential. In neuropathies such as demyelinating or dismyelinating diseases, chronic demyelination and defective remyelination occur repeatedly, leading to more severe neuropathy. As yet, little is known about the possibility of drug target-specific medicine for such diseases. In the developing peripheral nervous system (PNS), myelin sheaths form as Schwann cells wrap individual axons. It is thought that the development of a drug promoting myelination by Schwann cells would provide effective therapy against peripheral nerve disorders: to test such treatment, genetically modified mice overexpressing the drug target molecules are needed. We previously identified an Arf6 activator, the guanine-nucleotide exchange factor cytohesin-1, as the signaling molecule controlling myelination of peripheral axons by Schwann cells; yet, the important issue of whether cytohesin-1 itself promotes myelin thickness in vivo has remained unclear. Herein, we show that, in mouse PNS nerves, Schwann cell-specific expression of wild-type cytohesin-1 exhibits enhanced myelin thickness. Downstream activation of Arf6 is also seen in these transgenic mice, revealing the involvement of the cytohesin-1 and Arf6 signaling unit in promoting myelination. These results suggest that cytohesin-1 may be a candidate for the basis of a therapy for peripheral neuropathies through its enhancement of myelin thickness.

Purpose: We investigated the in vitro inhibitory effects of avanafil, a novel, potent inhibitor of phosphodiesterase-5, on 11 phosphodiesterases. We also studied its potentiation of penile tumescence in dogs., Materials and Methods: Phosphodiesterase assay was done with the 4 phosphodiesterase-5 inhibitors avanafil, sildenafil, vardenafil and tadalafil using 11 phosphodiesterase isozymes. In anesthetized dogs the pelvic nerve was repeatedly stimulated to evoke tumescence. Intracavernous pressure was measured after avanafil or sildenafil administration., Results: Avanafil specifically inhibited phosphodiesterase-5 activity at a 50% inhibitory concentration of 5.2 nM. Avanafil showed higher selectivity (121-fold) against phosphodiesterase-6 than sildenafil and vardenafil (16 to 21-fold) and showed excellent selectivity (greater than 10,000-fold) against phosphodiesterase-1 compared with sildenafil (375-fold). Avanafil also had higher selectivity against phosphodiesterase-11 than tadalafil (greater than 19,000 vs 25-fold). Avanafil also showed excellent selectivity against all other phosphodiesterases. After intravenous administration in anesthetized dogs the 200% effective dose of avanafil and sildenafil on the penile tumescence was 37.5 and 34.6 μg/kg, respectively. After intraduodenal administration the 200% effective dose of avanafil and sildenafil on tumescence was 151.7 and 79.0 μg/kg at the peak time, respectively. Time to peak response with avanafil and sildenafil was 10 and 30 minutes, respectively, indicating a more rapid onset of avanafil., Conclusions: Avanafil has a favorable phosphodiesterase-5 selectivity profile compared to that of marketed phosphodiesterase-5 inhibitors. Avanafil shows excellent in vitro and in vivo potency, and fast onset of action for penile erection. Cumulative data suggest that avanafil has a promising pharmacological profile for erectile dysfunction., (Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Introduction: Phosphodiesterase type 5 (PDE5) inhibitors are indicated for the treatment of erectile dysfunction (ED); however, they can also inhibit other PDE isozymes, affecting their target tissues (e.g., PDE1: heart; PDE6: retina; and PDE11: skeletal muscle), which in some cases can cause unwanted side effects and therapy discontinuation. Data from in vitro studies showed that avanafil, a PDE5 inhibitor for the treatment of ED, exhibited strong selectivity toward PDE5 and against all other PDE isozymes., Aim: To review the inhibitory effects of avanafil for PDE isozymes compared with those of sildenafil, tadalafil, and vardenafil and to discuss these results within the context of clinical trial safety observations., Methods: Review of in vitro selectivity data for avanafil (published primary data from a peer-reviewed journal and scientific congress abstracts); PubMed search for pertinent publications on PDE5 inhibitor safety data; and review of published articles and abstracts from avanafil phase 1, 2, and 3 clinical trials., Main Outcome Measures: A low incidence of some PDE-related adverse events may be reflected by the high selectivity of avanafil against non-PDE5 isozymes., Results: Avanafil is highly selective toward PDE5 and against all other PDE isozymes tested. Lower selectivity against PDE1, PDE6, and PDE11 is consistent with results from randomized, placebo-controlled, phase 3 trials in which musculoskeletal and hemodynamic adverse events were reported in <2% of patients and no color vision-related abnormalities were reported with avanafil doses up to 200 mg once daily., Conclusions: Data suggest that avanafil may confer a safety benefit, in terms of a lower incidence of specific adverse events, by virtue of its high specificity to PDE5 and its overall selectivity against other PDE isozymes., (© 2012 International Society for Sexual Medicine.)

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.

Contraction and relaxation of vascular smooth muscle and cardiac myocytes are key physiological events in the cardiovascular system. These events are regulated by second messengers, cAMP and cGMP, in response to extracellular stimulants. The strength of signal transduction is controlled by intracellular cyclic nucleotide concentrations, which are determined by a balance in production and degradation of cAMP and cGMP. Degradation of cyclic nucleotides is catalyzed by 3',5'-cyclic nucleotide phosphodiesterases (PDEs), and therefore regulation of PDEs hydrolytic activity is important for modulation of cellular functions. Mammalian PDEs are composed of 21 genes and are categorized into 11 families based on sequence homology, enzymatic properties, and sensitivity to inhibitors. PDE families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, enzymatic regulation by phosphorylation and regulatory proteins, subcellular localization, and interaction with association proteins. Each unique variant is closely related to the regulation of a specific cellular signaling. Thus, multiple PDEs function as a particular modulator of each cardiovascular function and regulate physiological homeostasis.

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Phosphodiesterase (PDE) 7B, a cAMP-specific PDE which is dominantly expressed in striatum, is expected to be involved in dopaminergic signaling in striatal neurons. Here we show, for the first time, the involvement of the dopaminergic signaling pathway in transcriptional activation of rat PDE7B in primary striatal culture. RT-PCR analysis revealed that dopamine, D1 agonist, forskolin and 8-Br-cAMP stimulation potentiated PDE7B transcription in striatal neurons, while D2 agonist failed to activate the PDE7B transcription. Pre-treatment with D1 antagonist abolished the dopamine- or D1 agonist-induced transcriptional activation of PDE7B. The activation of PDE7B transcription by these stimulators was completely ablated by pre-treatment of the cells with a cAMP-dependent protein kinase inhibitor, H-89. RT-PCR using splice variant-specific primers revealed that transcription of PDE7B1, but not of other splice variants, was activated by D1 agonist. We determined the putative transcription start site of PDE7B1, a brain-specific splice variant of PDE7B, by 5'-RACE and identified a promoter region of PDE7B1. Sequence analysis of the PDE7B1 promoter revealed the presence of a canonical cAMP-response element at 166 bp upstream of the putative transcription start site. The cAMP-responsiveness of the PDE7B1 promoter was demonstrated by functional promoter analysis using the luciferase reporter system. Deletion and mutation of the cAMP-response element site in the PDE7B1 promoter abolished the forskolin-induced activation of the PDE7B1 promoter activity. Electrophoretic mobility shift assay showed the binding of cAMP-response element binding protein to the PDE7B1 promoter. These data demonstrate the dopamine D1 receptor-mediated transcriptional activation of PDE7B through the cAMP/cAMP-dependent protein kinase/cAMP-response element binding protein pathway in striatal neurons.

Our previous studies have suggested that two phosphodiesterase type 10A (PDE10A) variants, PDE10A1 and PDE10A2 transcripts, are mainly expressed in humans and that PDE10A2 and PDE10A3 transcripts are major variants in rats. In the present study, immunoblot analysis demonstrated that PDE10A proteins, especially PDE10A2, are more abundant in membrane fractions than in cytosolic fractions of rat striatum. Recombinant PDE10A1 and PDE10A3 were produced only in cytosolic fractions of transfected PC12h cells. By contrast, recombinant PDE10A2 was present mainly in membrane fractions. This finding agreed well with the result of subcellular fractionation of PDE10A in rat striatum. Immunocytochemical analysis showed that PDE10A2 was localized in the Golgi apparatus of transfected PC12h cells. PDE10A2 was phosphorylated by cAMP-dependent protein kinase (PKA) at Thr16. Interestingly, recombinant protein of wild-type PDE10A2, but not PDE10A2 mutant with an Ala replacement at Thr16, was distributed to cytosolic fractions by co-transfection with a plasmid encoding the catalytic subunit of PKA. A PDE10A2 mutant with Glu substitution at Thr16, which can be a mimic of phosphorylation, was localized in the cytosolic fractions of transfected PC12h cells. These observations implied that phosphorylation of PDE10A2 at Thr16 by PKA caused alteration of subcellular localization of PDE10A2 from the Golgi apparatus to cytosol. It is hypothesized that cAMP signaling in the Golgi area and the cytosol in neurons is controlled through alteration of subcellular localization of PDE10A brought by activation of PKA in response to intracellular elevations of cAMP.

Phosphodiesterase-5 (PDE5) and cGMP-dependent protein kinase (PKG) play key roles in cGMP signaling. PDE5 has a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that binds cGMP at allosteric sites. We recently demonstrated that in corpus cavernosum, PDE5 concentration exceeds basal cGMP by ~5-fold making it possible that its allosteric sites could bind a significant fraction of the total cellular cGMP. It is hypothesized that the allosteric sites regulate cGMP signaling by sequestering cGMP. At 60 nM cGMP in vitro, which approaches a stimulated concentration of cGMP in rabbit corpus cavernosum, isolated R domain inhibits both cGMP hydrolysis by C domain and activation of PKG (IC50 values of 388 and 100 nM, respectively). Prior phosphorylation of R domain by cyclic nucleotide-dependent protein kinases, which increases its cGMP-binding affinity, also increases its potency for inhibiting both cGMP hydrolysis by C domain and cGMP activation of PKG (IC50 values of 58 and 38 nM, respectively). In rabbit corpus cavernosum, PDE5 concentration (94 nM) exceeds these values. These findings support our hypothesis that physiological concentrations of R domain regulate cGMP signaling by sequestering this nucleotide and that phosphorylation of R domain modulates this effect. This could provide for negative feedback control of cGMP-signaling.

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions., (Copyright 2003 Wiley-Liss, Inc.)

Cyclic nucleotide phosphodiestease (PDE) type 8 is categorized into a family of 3-isobutyl-1-methylxanthine-insensitive PDE hydrolyzing cAMP with high affinity. We have isolated cDNAs encoding rat PDE8A and PDE8B from brain and testis, respectively. The sequence analysis demonstrated that rat PDE8A was a protein of 823 amino acid residues. Rat PDE8B protein was predicted as an N-terminal truncated form of 760 amino acid residues. Both of rat PDE8 proteins include REC, PAS and catalytic PDE domains. Tissue-specific expression patterns of rat PDE8A and PDE8B transcripts were demonstrated by Northern blot analysis. Rat PDE8A transcripts were rich in the liver and testis, and those of rat PDE8B were particularly abundant in the brain and were not expressed in the thyroid gland, while human thyroid gland contains PDE8B transcripts at a high level. Rat PDE8B transcripts were found in all brain regions other than cerebellum and shown to exist in the neuronal cells in in situ hybridization. Mouse PDE8B1 sequence was also identified by a database search and sequence alignment, revealing a protein of 885 amino acid residues, which is 99% and 96% identical to rat and human PDE8B1, respectively. As well as rat PDE8B, expression of mouse PDE8B transcripts was not confined to the thyroid gland. Species-dependent tissue expression pattern was quite unique features of PDE8B.

Novel 1,7- and 2,7-naphthyridine derivatives, designed by the introduction of nitrogen atom into the phenyl ring of previously reported 4-aryl-1-isoquinolinone derivatives, were disclosed as a new structural class of potent and specific PDE5 inhibitors. Among them, 2,7-naphthyridine 4c showed potent PDE5 inhibition (IC(50)=0.23 nM) and one of the best PDE5 specificities against PDEs1-4,6 (>100,000-fold selective versus PDE1-4, 240-fold selective vs PDE6). This compound showed more potent relaxant effects on isolated rabbit corpus cavernosum (EC(30)=5.0 nM) than Sildenafil (EC(30)=8.7 nM). The compound 4c (T-0156) was selected for further biological and pharmacological evaluation of erectile dysfunction.

The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Ialpha or PKG-Ibeta on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Ialpha strongly inhibited cGMP hydrolysis with an IC(50) value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously. By contrast, PKG-Ibeta, which has lower affinity for cGMP than does PKG-Ialpha, inhibited cGMP hydrolysis with an IC(50) of approx. 1 microM. Inhibition by PKG-Ialpha was more effective than that by PKG-Ibeta, consistent with their relative affinities for cGMP. Autophosphorylation of PKGs increased their cGMP-binding affinities and their inhibitory effects on PDE5 hydrolysis of cGMP. Autophosphorylation of PKG-Ibeta increased its inhibitory potency on PDE5 hydrolysis of cGMP by 10-fold compared with a 2-fold increase upon autophosphorylation of PKG-Ialpha. The results indicate that cGMP bound to allosteric cGMP-binding sites of PKG is protected from hydrolysis by PDE5 and that persistent protection of cGMP by either non-phosphorylated or autophosphorylated PKGs may be a positive-feedback control to sustain cGMP signalling.

Full-length cDNAs of human cyclic nucleotide phosphodiesterase 8B (PDE8B) were isolated. Enzymatic characteristics of a dominant variant encoding a protein of 885 residues (PDE8B1) were compared with those of PDE8A1. The recombinant PDE8A1 and PDE8B1 proteins of an entire form were produced in both cytosolic and membrane fractions of the transfected COS cells. The human PDE8B1 was a high-affinity cAMP-PDE with K(m) value of 101+/-12 nM for cAMP, which is greater than that of PDE8A1 (40+/-1 nM). Relative V(max) value of PDE8A1 was 57+/-8% compared with that of PDE8B1 (100+/-12%). Although PDE8A1 was moderately inhibited by dipyridamole with IC(50) value of 8+/-2 microM, the compound antagonized the PDE8B1 activity at three-fold higher concentration (IC(50)=23+/-2 microM). The human PDE8B gene was composed of 22 exons, spanning over 217 kb. Although overall sequence identity between PDE8A1 and PDE8B1 was 68%, positions of junctions of each exon between the PDE8A1 and PDE8B1 sequences were well matched, indicating evolutionary relatedness of both genes.

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [3H]Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1-methylxanthine, EDTA, or cGMP, but not by cAMP or 5'-GMP. PDE5 was the only [3H]sildenafil binding protein detected in human lung extract. Using purified recombinant PDE5, [3H]sildenafil exchange dissociation yielded two components with t1/2 values of 1 and 14 min and corresponding calculated KD values of 12 and 0.83 nM, respectively. This implied the existence of two conformers of the PDE5 catalytic site. [3H]Sildenafil binding isotherm of PDE5 indicated KD was 8.3 to 13.3 nM, and low cGMP decreased the KD to 4.8 nM but only slightly increased Bmax to a maximum of 0.61 mol/mol-subunit. Results suggest that these effects occur via cGMP binding to the allosteric cGMP binding sites of PDE5. Results imply that by inhibiting PDE5 and thereby increasing cGMP, sildenafil accentuates its own binding affinity for PDE5, which further elevates cGMP. The data also indicate that after physiological elevation, cGMP may directly stimulate the catalytic site by binding to the allosteric cGMP-binding sites of PDE5, thus causing negative feedback on this pathway.

Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Delta(1-52)PKG-I beta). On saturation with cGMP, the radius of gyration of Delta(1-52)PKG-I beta increases from 29.4 +/- 0.1 A to 40.1 +/- 0.7 A, and the maximum linear dimension increases from 90 A +/- 10% to 130 A +/- 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to Delta(1-52)PKG-I beta indicates that elongation of Delta(1-52)PKG-I beta requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of Delta(1-52)PKG-I beta. We also predict that there is little or no cooperativity in cGMP binding to the two sites of Delta(1-52)PKG-I beta under the conditions used here. Results obtained by using the Delta(1-52)PKG-I beta monomer indicate that a previously observed elongation of PKG-I alpha is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-I beta activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.

The enzymological and pharmacological properties of 2-(2-Methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), a new phosphodiesterase type 5 inhibitor, were studied in vitro and in vivo. The inhibitory effects of T-0156 on six phosphodiesterase isozymes isolated from canine tissues were investigated. T-0156 specifically inhibited the hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5, at low concentration (IC(50)=0.23 nM), in a competitive manner. T-0156 also inhibited phosphodiesterase type 6 with IC(50) value of 56 nM, which was 240-fold higher than that for inhibition of phosphodiesterase type 5. T-0156 had low potencies against phosphodiesterase types 1, 2, 3, and 4 (IC(50)>10 microM). In the isolated rabbit corpus cavernosum, T-0156 at 10 and 100 nM increased cGMP levels (100 nM T-0156-treated: 6.0+/-1.5 pmol/mg protein, vehicle-treated: 1.1+/-0.4 pmol/mg protein, P<0.05), causing relaxation of the tissue. T-0156 at 1 to 100 nM potentiated the electrical field stimulation-induced relaxation in the isolated rabbit corpus cavernosum in a concentration-dependent manner (100 nM T-0156-treated: 76.9+/-19.8%, vehicle-treated: 12.3+/-10.1%, P<0.05). Intraduodenal administration of T-0156 at 100 to 1000 microg/kg potentiated the pelvic nerve stimulation-induced tumescence in anesthetized dogs (1000 microg/kg T-0156-treated: 279.0+/-38.4%, vehicle-treated: 9.8+/-4.5%, P<0.05). These results suggested that T-0156 enhanced the nitric oxide (NO)/cGMP pathway, probably through blockade of phosphodiesterase type 5 in vitro and in vivo experimental conditions. The present study clearly showed that T-0156 is a potent and highly selective phosphodiesterase type 5 inhibitor, which is a useful tool for pharmacological studies in vitro and in vivo.

cDNA species coding for novel variants of cyclic-AMP-specific phosphodiesterases (PDEs), namely the PDE7B family, were isolated from rats and characterized. Rat PDE7B1 (RNPDE7B1) was composed of 446 amino acid residues. Rat PDE7B2 (RNPDE7B2) and PDE7B3 (RNPDE7B3), which possessed unique N-terminal sequences, consisted of 359 and 459 residues respectively. Northern hybridization analysis showed that rat PDE7B transcripts were particularly abundant in the striatum and testis. PCR analyses revealed that rat PDE7B2 transcripts were restricted to the testis and that low levels of PDE7B3 transcripts were expressed in the heart, lung and skeletal muscle. In situ hybridization analysis demonstrated that rat PDE7B transcripts were expressed in striatal neurons and spermatocytes. In spermatocytes, rat PDE7B transcripts were expressed in a stage-specific manner during spermatogenesis. The K(m) values of recombinant rat PDE7B1, PDE7B2 and PDE7B3 for cAMP were 0.05, 0.07 and 0.05 microM respectively. Each rat PDE7B variant was the most sensitive to 3-isobutyl-1-methylxanthine (IC(50) 1.5-2.1 microM). Two phosphorylation sites for cAMP-dependent protein kinase (PKA) were found in rat PDE7B1 and PDE7B3, whereas rat PDE7B2 possessed one site. PKA-dependent phosphorylation was observed in C-terminal phosphorylation sites of three rat PDE7B variants, in addition to unique N-terminal regions of rat PDE7B1 and PDE7B3. Unique tissue distribution and PKA-dependent phosphorylation of PDE7B variants suggested that each variant has a specific role for cellular functions via cAMP signalling in various tissues.

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1, 2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration (IC(50) = 1.0 nM, K(i) = 1.2 nM), in a competitive manner. In contrast, the IC(50) values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 microM. T-1032 also inhibited PDE6 from canine retina with an IC(50) of 28 nM, which is of the same order of magnitude as the IC(50) of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the N-terminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP- and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K(m) values of 3.0 and 1.4 microm, respectively, and the V(max) value with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K(m) values as PDE11A4, the relative V(max) values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products.

PDE10A is a cyclic nucleotide phosphodiesterase (PDE) exhibiting properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. The transcripts are specifically expressed in the striatum. The human gene encoding PDE10A was cloned and investigated. The PDE10A gene spanned > 200 kb and contained 24 exons. The exon-intron organization of PDE10A was different from those of PDE5A and PDE6B, although these three PDEs include two GAF domains and have similar amino-acid sequences. The promoter sequence of PDE10A was highly GC-rich and did not contain a TATA motif and a CAAT box, suggesting it is a housekeeping gene. In Caenorhabditis elegans, the C32E12.2 gene encoding a probable PDE that is 48% identical to the human PDE10A protein showed similar exon organization to PDE10A but not PDE5A and PDE6B. This, together with the phylogenic tree analysis, suggested that the ancestral gene for PDE10A existed in a lower organism such as C. elegans.

We isolated a human cAMP-specific phosphodiesterase (PDE7B) cDNA from human caudate nucleus. The human PDE7B was composed of 450 amino acid residues with a molecular mass of 51,835 Da. The deduced amino acid sequence of human PDE7B was 64.1% identical to that of human PDE7A (67.1% identity in the catalytic region). Northern blot analysis demonstrated that PDE7B transcripts were abundantly expressed in the putamen, caudate nucleus, and heart followed by skeletal muscle, pancreas, and occipital pole. Recombinant PDE7B expressed in transfected COS-7 cells had a low cAMP K(m) value of 0. 13 microM, which is similar to the K(m) value of recombinant human PDE7A expressed in transfected COS-7 cells. Interestingly, the relative V(max) value of recombinant PDE7B was half to one-third of recombinant PDE7A. The PDE7B activity was inhibited by dipyridamole and SCH51866, with IC(50) values of 1.1 microM and 1.5 microM, respectively. Thus, the PDE7B exhibited unique tissue distribution in humans and kinetic profiles. Human PDE7B showed the lowest K(m) values compared to the other cAMP-hydrolyzing PDEs which have been reported to be expressed in the brain. Therefore, human PDE7B may be involved in the control of cAMP-mediated neural activity and cAMP metabolism in the brain., (Copyright 2000 Academic Press.)

We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E. coli. This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum. Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats. Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas. Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues.

Guanosine 3',5'-cyclic monophosphate (cGMP) has been recently reported to be involved in bone formation. ATDC5 cells were used to investigate cGMP metabolism during chondrogenic differentiation. Natriuretic peptide receptor (NPR)-A and NPR-B coupled with guanylate cyclase (GC) mediate biological functions of NPs, whereas NPR-C uncoupled with GC is thought to be the clearance receptor for NPs. The amounts of NPR-A, NPR-B, and CNP transcripts were increased but the amount of NPR-C transcripts was decreased in association with the chondrogenic differentiation of ATDC5 cells. CNP, a specific ligand for NPR-B lets ATDC5 cells accumulate great amounts of cGMP, revealing NPR-B as a dominant biological receptor through differentiation. cGMP hydrolytic activities of PDE1 and PDE5 existed in ATDC5 cells, and the activity of PDE1, which is stimulated by Ca(2+) and calmodulin (CaM) was major of them. Total cGMP hydrolytic activities as well as the amounts of PDE1 and PDE5 transcripts were enhanced during chondrogenic differentiation. Therefore, cGMP production and hydrolysis, cGMP metabolism was considered to be activated in association with chondrogenic differentiation of ATDC5 cells. These observations may lead to a better understanding of cGMP in the chondrocytes where bone formation occurs.

PDE10A, a phosphodiesterase (PDE) exhibiting properties of a cAMP PDE and a cAMP-inhibited cGMP PDE, was cloned and investigated in detail in rats. PDE10A transcripts were abundant in the brain and testis. In situ hybridization analysis using a PDE10A riboprobe demonstrated the presence of PDE10A transcripts in the neurons of the striatum and the olfactory tubercle regions of the brain. Rat PDE10A cDNAs were isolated from a brain cDNA library and nucleotide sequence analysis revealed several N-terminal variants. The deduced amino-acid sequence of one of the major variant forms contained 794 amino acids, and it was 96% identical to that of the human PDE10A2. The other major form has a distinct N-terminal sequence that is not found in humans. PDE10A was partially purified from rat striatum and testis, and characterized with respect to Km, inhibitor sensitivity and immunoreactivity to an anti-PDE10A serum. These findings indicate that PDE10A functions in these tissues.