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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.

Phosphodiesterase (PDE) 10A is an attractive therapeutic target for schizophrenia. Here, we investigated the antipsychotic-like effects of a novel PDE10A inhibitor, 1-({2-(7-fluoro-3-methylquinoxalin-2-yl)-5-[(3R)-3-fluoropyrrolidin-1-yl]pyrazolo[1,5-α]pyrimidin-7-yl}amino)-2-methylpropan-2-ol hydrochloride (MT-3014) in rats. MT-3014 showed a potent and selective inhibitory effect against PDE10A (IC50 = 0.357 nmol/L). Oral administration of MT-3014 (1.0–10 mg/kg) significantly increased the levels of cAMP, cGMP and cAMP response element-binding protein (CREB) phosphorylation in the rat striatum. MT-3014 decreased MK-801 (0.075 mg/kg)-induced hyperactivity (ED50 = 0.30 mg/kg) in a dose-dependent manner, although it decreased spontaneous locomotion in control rats (ED50 = 0.48 mg/kg); its effects were equivalent to those of risperidone. MT-3014 (0.3–3.0 mg/kg and 0.2 mg/kg) attenuated MK-801-induced prepulse inhibition deficits and cognitive deficits in rats, respectively, whereas risperidone attenuated MK-801-induced prepulse inhibition at only a high dose and failed to improve MK-801-induced cognitive deficits. Similar to risperidone (ID50 = 0.63 mg/kg), MT-3014 suppressed the conditioned avoidance response (ID50 = 0.32 mg/kg). Interestingly, MT-3014 did not elicit catalepsy and plasma prolactin increases at high doses. Furthermore, it also did not affect body weight. A positron emission tomography study using [11C]IMA107 showed a plasma concentration-dependent increase in brain PDE10A occupancy after oral administration of MT-3014 within the pharmacological dose range in rats. Brain PDE10A occupancy corresponding to the ID50 value in the conditioned avoidance response was approximately 60%, predicting the target occupancy in patients with schizophrenia. These results suggest that MT-3014 may be a novel antipsychotic drug, which is expected to have additional effects on cognitive impairment, without the prominent side effects associated with current atypical antipsychotics.

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.

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.

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.

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.

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.

cDNA encoding a novel phosphodiesterase (PDE) was isolated from a human fetal lung cDNA library and designated PDE10A. The deduced amino acid sequence contains 779 amino acids, including a putative cGMP binding sequence in the amino-terminal portion of the molecule and a catalytic domain that is 16-47% identical in amino acid sequence to those of other PDE families. Recombinant PDE10A transfected and expressed in COS-7 cells hydrolyzed cAMP and cGMP with Km values of 0.26 and 7.2 microM, respectively, and Vmax with cGMP was almost twice that with cAMP. Of the PDE inhibitors tested, dipyridamole was most effective, with IC50 values of 1.2 and 0.45 microM for inhibition of cAMP and cGMP hydrolysis, respectively. cGMP inhibited hydrolysis of cAMP, and cAMP inhibited cGMP hydrolysis with IC50 values of 14 and 0.39 microM, respectively. Thus, PDE10A exhibited properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. PDE10A transcripts were particularly abundant in the putamen and caudate nucleus regions of brain and in thyroid and testis, and in much lower amounts in other tissues. The PDE10A gene was located on chromosome 6q26 by fluorescent in situ hybridization analysis. PDE10A represents a new member of the PDE superfamily, exhibiting unique kinetic properties and inhibitor sensitivity.

After our recent findings that the amino-terminal portion of rat cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) differs from those of bovine and human cGB-PDEs, we found two forms of canine cGB-PDE cDNAs (CFPDE5A1 and CFPDE5A2) in canine lung. Each contained a distinct amino-terminal sequence, CFPDE5A1, possessing an amino-terminal portion with sequence similar to those of bovine and human, and CFPDE5A2, having one similar to that of rat. Other portions coding for the cGMP binding domains and the catalytic domain were conserved. Both CFPDE5A1 and CFPDE5A2 transcripts were detected in the cerebellum, hippocampus, retina, lung, heart, spleen, and thoracic artery. CFPDE5A1 transcripts were particularly abundant in the pylorus, whereas CFPDE5A2 transcripts were quite low in this tissue. CFPDE5A1 and CFPDE5A2 expressed in COS-7 cells had cGMP K mvalues of 2.68 and 1.97 μm, respectively, and both were inhibited by a low concentration of a cGB-PDE inhibitor, Zaprinast. Both CFPDE5A1 and CFPDE5A2 bound cGMP to their allosteric cGMP binding domains, and this cGMP binding was stimulated by 3-isobutyl-1-methylxanthine. Thus, two types of alternative splice variants of canine cGB-PDE have been identified and shown to have similar biological properties in vitro.

Phenotypic modulation of vascular smooth muscle cells (SMCs) plays a central role in the pathogenesis of atherosclerosis. Natriuretic peptide receptor-C (NPR-C) is highly expressed in vascular SMCs in the experimental arteriosclerotic neointimal area as well as in cultured SMCs, suggesting that increased expression of the NPR-C gene is related to the phenotypic alteration of vascular SMCs. To elucidate the molecular mechanisms and to identify the essential DNA sequences in NPR-C gene expression, a genomic clone containing over 8 kilobases of the 5'-flanking region of the human NPR-C gene has been isolated. Sequence analysis revealed that a number of putative regulatory elements including unusual tandem repeated AP-2-like sequences were observed in the 5'-flanking region. Primer extension and ribonuclease protection analyses revealed that transcription of the human NPR-C gene starts from two major regions. Promoter analysis using deletion constructs in human cells, highly producing NPR-C transcripts, showing that the region (from - 33 to + 13 relative to the transcription start point) had a potential promoter activity suggested that the region from -33 to + 13, containing a pyrimidine-rich stretch composed of four CTTTTT-repeated sequences, is sufficient for the proximal promoter activity. Moreover, three distinct DNA sequences surrounding the transcription start site (P1, from -60 to -33; P2, from + 14 to +40; P3, from +41 to +66) were revealed to be functional as a cis-acting positive enhancer, and a nuclear protein(s) from the human cells was demonstrated to specifically bind to the sequences, respectively. However, promoter analysis has shown that the P2 and P3 sequences could not activate the human NPR-C promoter in a synergistic manner. On the basis of deoxyribonuclease I footprinting analysis showing that a DNA element from +48 to +60 within the P3 sequence is preferentially protected, the P3 sequence appears to contain a potential regulatory element involved in NPR-C gene expression. The present study demonstrated the structure of the 5'-regulatory region of the human NPR-C gene and multiple cis-acting positive sequences closely located around the transcription start points with an important role in regulation of human NPR-C gene expression.

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.

A full-length cDNA, encoding the mouse atrial natriuretic peptide clearance receptor (ANP-CR), was isolated from a mouse lung cDNA library. The deduced amino acid sequence of the mouse ANP-CR, showing a typical tripartite organization which lacks a guanylyl cyclase domain, was extremely well conserved compared with the ANP-CR homologs. To understand the molecular mechanisms underlying the regulation of mouse ANP-CR gene expression and to define the essential DNA sequences for the transcriptional activity, a genomic clone containing over 9 kb of the 5'-flanking region of the mouse ANP-CR gene has been isolated from a mouse genomic library. Sequence analysis revealed that the 2.3-kb region upstream from an ATG codon of the mouse ANP-CR gene contained a number of putative regulatory elements; TATA box, CAAT box, cAMP response element, AP-1 and two shear stress responsive elements. Additionally, an unusual feature was the presence of the tandem-repeated AP-2-like elements, which were closely overlapped with SP-1 element. Promoter analysis using deletion plasmids in mouse Balb/3T3 cells, highly producing ANP-CR mRNA, demonstrated that deletion of the sequence from -144 to +46 relative to the transcription start point caused a dramatic decrease of the transcriptional activity and that the TATA box at -269 was not essential for the basal transcriptional activity. Primer extension analysis indicated that transcription of the mouse ANP-CR gene starts from at least two major sites, suggesting that the sequence from -144 to +46, which was shown to involve a novel sequence composed of tandem-repeated TATA-box-like elements, contained promoter sequences. Furthermore, cis-acting negative elements were shown to be situated in three regions (from -1178 to -708, from -707 to -625 and from -248 to -145) of the mouse ANP-CR gene promoter.

The monitor peptide receptor in the small intestine was investigated. The receptor is on the intestinal mucosal cells [Biochem. J., in press]. The specific binding of the (125)I labeled monitor peptide to dispersed rat small intestinal cells was inhibited by treatment with p-toluensulfonyl-L-lysine chloromethyl ketone (TLCK), an affinity labeling reagent for trypsin. Soybean trypsin inhibitor (SBTI) did not inhibit the binding. Analysis with reduced SDS electrophoresis-autoradiography indicated that an affinity-cross-linked complex of the (125)I labeled monitor peptide and the receptor was abolished by the TLCK treatment but was not affected by the presence of SBTI. Histochemical studies found a predominatnt binding of (125)I labeled monitor peptide on cholecystokinin (CCK)-immunoreactive cells in tissue sections of the rat upper intestine. The result suggests that the monitor peptide receptor is on the surface of CCK-producing cells.

Publisher Summary This chapter describes the primary structure, enzymatic characteristics, inhibitor sensitivity, and gene expression of PDE9A, a cGMP-specific PDE with the lowest Km value among all PDEs. Several intracellular small molecules, including cyclic nucleotides (cAMP and cGMP) and ions, act as second messengers to regulate various cellular functions. For instance, cGMP, which is produced by extracellular molecules via activation of guanylyl cyclases, activates cGMP-dependent protein kinases (PKG) and cGMP-gated ion channels. This activation can lead to relaxation of vascular smooth muscle, airway distension, inhibition of cell proliferation, inhibition of platelet aggregation, neuronal transmission, visual signaling, and apoptosis. Cyclic nucleotide phosphodiesterases (PDEs), which comprise 11 families classified on the basis of amino acid sequence homology and substrate specificity, are known to hydrolyze cyclic nucleotides. PDE9A inhibitors improve long-term potentiation in several animal models of cognition. Specific inhibitors of each PDE are used as drugs to treat a variety of diseases. For instance, PDE5 inhibitors, such as sildenafil, vardenafil, tadalafil, and udenafil, are prescribed for erectile dysfunction. On the other hand, cilostazol, a PDE3 inhibitor, is used to treat intermittent claudication. Several PDE4 inhibitors are also being developed for treatment of allergies, including asthma and chronic obstructive pulmonary disease. Thus, PDE families are considered to be drug targets, and many pharmaceutical companies are developing new PDE inhibitors.

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.

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.

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 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

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.

SUMMARY 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 Ca2+ 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.

We have isolated a novel alternative splice variant of human cAMP- and cGMP-hydrolyzing phosphodiesterase (PDE10A2) from human fetal lung. The N-terminal sequence of human PDE10A2 differed from that of human PDE10A1 reported previously. PDE10A1 and PDE10A2 expressed in COS-7 cells have cGMP K(m) values of 14 and 13 microM, low cAMP K(m) values of 0.28 and 0.22 microM, and high cAMP K(m) values of 0.96 and 1.1 microM, respectively, at high concentrations of cGMP and cAMP. PCR analysis demonstrated that both PDE10A1 and PDE10A2 transcripts are present in various human tissues and that PDE10A2 transcripts are a major form in some human tissues. The unique N-terminus of PDE10A2 has a putative phosphorylation site by cAMP-dependent protein kinase (cAK), but PDE10A1 does not. The recombinant PDE10A2 protein is preferentially phosphorylated by cAK, although the recombinant PDE10A1 protein is not phosphorylated by cAK.

We have reported alternative splice variants of cGMP-binding cGMP-specific phosphodiesterases (PDE5A), i.e. rat PDE5A2, human PDE5A1, canine PDE5A1 and PDE5A2, which possess distinct N-terminal sequences. In this study, the DNA sequences corresponding to the unique N-terminal portions of PDE5A1 and PDE5A2 were shown to be tandemly located upstream of exons encoding the common region of PDE5A in both human and rat PDE5A genes. The presence of human PDE5A2 and rat PDE5A1 transcripts in lung was confirmed by reverse transcriptase-PCR. These results indicated that two variant forms of PDE5A exist in humans, canines and rats. We examined the tissue distribution of the two variants of human PDE5A in adult and fetal humans. The patterns of expression of the two alternatively spliced transcripts of human PDE5A in human tissues differed. Many putative regulatory elements including cAMP response elements were observed in the 5'-untranslated region and intron of the PDE5A gene. The levels of the PDE5A transcripts, especially the PDE5A2 transcripts, were increased by a cAMP analogue in cultured rat vascular smooth muscle cells, indicating that the PDE5A2 is an inducible variant of PDE5A in rats.

1-Arylnaphthalene lignan, which had been reported as a PDE4 inhibitor by Iwasaki, was disclosed as a new structural class of PDE5 inhibitors. The structural requirements for potent and specific PDE5 inhibition were revealed in a 1-arylnaphthalene lignan series, in which 1-(3-bromo-4, 5-dimethoxyphenyl)-5-chloro-3-[4-(2-hydroxyethyl)-1-piperazinylcarbon yl]-2-(methoxycarbonyl)naphthalene hydrochloride (27q) showed the most potent and specific inhibition (PDE5 inhibition IC50 = 6.2 nM, selectivity for PDE5 against PDE1, -2, -3, and -416 000). It is noteworthy that 27q has the best selectivities against PDE isoforms among PDE5 inhibitors so far reported. Compound 27q exhibited almost the same relaxant effects on rat aortic rings as sodium 1-[6-chloro-4-[(3, 4-methylenedioxybenzyl)amino]quinazolin-2-yl]piperidine-4-ca rboxylate (35) (27q, EC50 = 0.10 microM; 35, EC50 = 0.20 microM) and was selected for further biological evaluation.

cGMP-binding, cGMP-specific phosphodiesterase which is encoded by the PDE5A gene plays important roles in cardiovascular system, and is a significant target molecule of therapeutic agents. However, little is known about molecular characteristics of the human PDE5A gene. The 4.4-kb cDNA encoding human PDE5A was isolated from lung and placenta cDNA libraries. The deduced amino acid sequence analysis demonstrated that N-terminal amino acid sequence is dissimilar to that of rat PDE5A [Kotera, J., Yanaka, N., Fujishige, K., Imai, Y., Akatsuka, H., Ishizuka, T., Kawashima, K. & Omori, K. (1997) Eur. J. Biochem. 249, 434-442]. Human PDE5A mRNA is produced in high amounts in various tissues such as pancreas, skeletal muscle, placenta, heart, thyroid, adrenal cortex, testis, small intestine and stomach. In addition, the megakaryocyte-like cell line Dami cells and two types of human vascular smooth muscle cells also produce the mRNA. Over 100-kb chromosomal DNA corresponding to the human PDE5A gene was isolated and analyzed. The human PDE5A gene was revealed to contain 21 exons. Comparison of genomic organization with the rod photoreceptor phosphodiesterase beta-subunit gene (PDE6B), which is another kind of cGMP-specific phosphodiesterase, has shown that the PDE5A and PDE6B genes are very similar in their relative exon intron organization. In particular, the evolutionary relatedness of these genes was suggested in the catalytic domain. Furthermore, chromosomal location of the PDE5A gene was defined as being chromosome 4q26 by fluorescent in situ hybridization analysis.

The cDNA encoding rat cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) was isolated from a rat lung cDNA library. Although the deduced amino acid sequence showed 93.4% similarity with that of bovine cGB-PDE, the N-terminal portion of rat cGB-PDE was extremely different from that of bovine. Northern blot analysis indicated that cGB-PDE transcripts in rats were expressed not only in aorta and lung, but also in several other tissues including cerebellum. In situ hybridization analysis demonstrated that cerebellar expression of cGB-PDE was confined to Purkinje cell layers in adult rats. To clarify the role of cGB-PDE in the cerebellum, we investigated expression of cGB-PDE mRNA in rats of various ages. cGB-PDE mRNA was not observed in the cerebellum of newborn rats, but levels of a cGB-PDE mRNA were markedly increased between 4 days and 28 days of age and reached a maximum in eight-week-old rats. In this study, we suggest that cGB-PDE plays important roles not only in regulating the relaxation of vascular vessels, but also in establishing neuronal networks in the cerebellum at an early postnatal stage. In addition the NO/cGMP/cGB-PDE pathway appears to be essential for the induction of long-term depression.

Linoleic acid and oleic acid markedly increased the influx of 45Ca into isolated intestinal epithelial cells, and this increase reflected a rise in the intracellular calcium level. Methyl linoleate had no effect, while glutamic acid and somatostatin both inhibited the linoleic acid-induced influx of 45Ca. In addition, methyl linoleate had no effect, while glutamic acid inhibited linoleic acid-induced hormone-responsive pancreatic exocrine secretion.

A specific binding of the cholecystokinin (CCK)-releasing peptide (monitor peptide) to isolated rat jejunal mucosal cells was investigated. The 125I-labelled purified monitor peptide bound to the rat jejunal cells, and a large excess amount of the non-labelled monitor peptide inhibited the binding. The binding was completed within 60 min at 37 degrees C. The optimum pH for the binding was 8-9. A Scatchard plot of the specific binding was linear, and the dissociation constant was 50 nM. The density of the monitor-peptide-binding sites was high in duodenum but low in ileal and absent in colonic mucosa. A recombinant monitor peptide and four kinds of point mutants of it were prepared. The binding of the mutant monitor peptides to the cells indicated that only a trypsin inhibitor of the mutants could bind to the mucosal cells. Human pancreatic secretory trypsin inhibitor inhibited the specific binding, but other trypsin inhibitors, i.e. bovine basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, egg-white trypsin inhibitor, leupeptin, antipain and FOY-305, did not affect the specific binding at all. These findings suggested that the specific binding site for the monitor peptide on the jejunal mucosal cells has a trypsin-like specificity, exhibiting a special specificity for the pancreatic-secretory-trypsin-inhibitor family. Autoradiography of an affinity-cross-linked complex of the 125I-labelled intact monitor peptide and the binding site suggested that its molecular mass was 33 kDa or 53 kDa in the presence or absence of 2-mercaptoethanol respectively.