Kotomi Fujishige

Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A).

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 K m values of 0.26 and 7.2 μm, respectively, and V max 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 μm 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 μm, 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 fluorescentin situ hybridization analysis. PDE10A represents a new member of the PDE superfamily, exhibiting unique kinetic properties and inhibitor sensitivity.

Isolation and Characterization of Two Novel Phosphodiesterase PDE11A Variants Showing Unique Structure and Tissue-specific Expression

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 μm, respectively, and the V maxvalue with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K m values as PDE11A4, the relativeV 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.

Immunohistochemical Localization of cGMP-binding cGMP-specific Phosphodiesterase (PDE5) in Rat Tissues

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.

Novel Alternative Splice Variants of cGMP-binding cGMP-specific Phosphodiesterase

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 m values 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.

Characterization and effects of methyl-2- (4-aminophenyl)-1,2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5)

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.

Enzymological and pharmacological profile of T-0156, a potent and selective phosphodiesterase type 5 inhibitor

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.

1,7- and 2,7-naphthyridine derivatives as potent and highly specific PDE5 inhibitors

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.

Localization of clearance receptor in rat lung and trachea: association with chondrogenic differentiation

The lung is rich in atrial natriuretic peptide binding sites, and the majority of them are considered to be the natriuretic peptide clearance receptor (NPR-C). In this study, localization of NPR-C in the rat lung and trachea was investigated by immunohistochemical analysis with the specific antibody. Positive staining was observed in the epithelial cell layers of the trachea and bronchiole and the myocardium surrounding the pulmonary vein. Moreover, expression of NPR-C was seen in mesenchymal cells; it was especially strong in cells in the perichondrium and decreased in chondrocytes in the cartilage. Because mesenchymal cells in the perichondrium differentiate to chondrocytes, NPR-C expression is suggested to be associated with chondrogenic differentiation. The chondrogenic cell line ATDC5 was used to study NPR-C expression during chondrogenic differentiation in vitro. The undifferentiated ATDC5 cells expressed NPR-C at a much higher level than the differentiated ATDC5 cells, in accordance with the observation of the immunohistochemical analysis in the cartilage. These findings suggest that NPR-C expression is differentially regulated in chondrocytes and that the natriuretic peptides may play a role in regulating chondrocyte development in the lung.The lung is rich in atrial natriuretic peptide binding sites, and the majority of them are considered to be the natriuretic peptide clearance receptor (NPR-C). In this study, localization of NPR-C in the rat lung and trachea was investigated by immunohistochemical analysis with the specific antibody. Positive staining was observed in the epithelial cell layers of the trachea and bronchiole and the myocardium surrounding the pulmonary vein. Moreover, expression of NPR-C was seen in mesenchymal cells; it was especially strong in cells in the perichondrium and decreased in chondrocytes in the cartilage. Because mesenchymal cells in the perichondrium differentiate to chondrocytes, NPR-C expression is suggested to be associated with chondrogenic differentiation. The chondrogenic cell line ATDC5 was used to study NPR-C expression during chondrogenic differentiation in vitro. The undifferentiated ATDC5 cells expressed NPR-C at a much higher level than the differentiated ATDC5 cells, in accordance with the observation of the immunohistochemical analysis in the cartilage. These findings suggest that NPR-C expression is differentially regulated in chondrocytes and that the natriuretic peptides may play a role in regulating chondrocyte development in the lung.