Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we report the cloning, expression, and characterization of a cAMP-specific phosphodiesterase (PDE) from Trypanosoma brucei (TbPDE2B). Using a bioinformatic approach, two different expressed sequence tag clones were identified and used to isolate the complete sequence of two identical PDE genes arranged in tandem. Each gene consists of 2,793 bases that predict a protein of 930 aa with a molecular mass of 103.2 kDa. Two GAF (for cGMP binding and stimulated PDEs, Anabaena adenylyl cyclases, and Escherichia coli FhlA) domains, similar to those contained in many signaling molecules including mammalian PDE2, PDE5, PDE6, PDE10, and PDE11, were located N-terminal to a consensus PDE catalytic domain. The catalytic domain is homologous to the catalytic domain of all 11 mammalian PDEs, the Dictyostelium discoideum RegA, and a probable PDE from Caenorhabditis elegans. It is most similar to the T. brucei PDE2A (89% identity). TbPDE2B has substrate specificity for cAMP with a K(m) of 2.4 microM. cGMP is not hydrolyzed by TbPDE2B nor does this cyclic nucleotide modulate cAMP PDE activity. The nonselective PDE inhibitors 3-isobutyl-1-methylxanthine, papaverine and pentoxifyline are poor inhibitors of TbPDE2B. Similarly, PDE inhibitors selective for the mammalian PDE families 2, 3, 5, and 6 (erythro-9-[3-(2-hydroxynonyl)]-adenine, enoximone, zaprinast, and sildenafil) were also unable to inhibit this enzyme. However, dipyridamole was a reasonably good inhibitor of this enzyme with an IC50 of 27 microM. cAMP plays key roles in cell growth and differentiation in this parasite, and PDEs are responsible for the hydrolysis of this important second messenger. Therefore, parasite PDEs, including this one, have the potential to be attractive targets for selective drug design.
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PMID:Cloning and characterization of a cAMP-specific phosphodiesterase (TbPDE2B) from Trypanosoma brucei. 1193 17

Cyclic nucleotides are involved in the control of pulmonary vascular tone. In the present study, we measured the cyclic nucleotide specific phosphodiesterase (PDE) activity in the media of bovine isolated main pulmonary artery (MPA). Total cAMP- and cGMP-PDE activities were measured in microsomal and cytosolic fractions. Both cyclic nucleotides were hydrolysed in these subcellular fractions at consistently higher rate in the cytosolic than in the microsomal fraction. Using different classes of PDE modulator, at least four PDE isoforms (PDE1, 3, 4 and 5) were identified in these fractions. PDE3 (cilostamide-sensitive), PDE4 (rolipram-sensitive) and PDE5 (zaprinast- and DMPPO-sensitive) isoforms appeared as the main isozymes implicated in the cAMP and cGMP hydrolytic activities. Calcium-camodulin stimulated PDE activity (PDE1) was mainly present in the cytosolic fraction. PDE2, although present, had a lower hydrolytic activity since addition of its specific inhibitor, erythro-9-(2-hydroxy-3nonyl)adenine (EHNA), to a combination of inhibitors of PDE3, 4 and 5 produced no further significant reduction in the enzymatic activity. Resolution of PDE activities from the cytosolic fraction using anion exchange chromatography confirmed this finding. Functional experiments performed in endothelium-denuded rings of rat MPA revealed that all specific PDE inhibitors used relaxed precontracted vascular smooth muscle preparations in a concentration-dependent manner. The rank order of potency was cilostamide >zaprinast>rolipram>>EHNA. The present study demonstrates the presence in the smooth muscle cells-containing layer of MPA of PDE1, 3, 4 and 5 isoforms and suggests that PDE3, 4 and 5 are the main enzymes involved in the control of vascular tone.
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PMID:Characterisation of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery. 1200 79

Therapies that mitigate the fibrotic process may be able to slow progressive loss of function in many lung diseases. Because cyclic adenosine monophosphate is known to regulate fibroblasts, the current study was designed to evaluate the activity of selective phosphodiesterase (PDE) inhibitors on two in vitro fibroblast responses: chemotaxis and contraction of three-dimensional collagen gels. Selective PDE4 inhibitors, rolipram and cilomilast, each inhibited the chemotaxis of human fetal lung fibroblasts (HFL-1) toward fibronectin in the blindwell assay system (control: 100% versus cilomilast [10 microM]: 40.5 +/- 7.3% versus rolipram: [10 microM] 32.1 +/- 2.7% cells/5 high-power fields; P < 0.05, both comparisons). These PDE4 inhibitors also inhibited contraction of three-dimensional collagen gels (control: 100% versus cilomilast: 167.7 +/- 6.9% versus rolipram: 129.9 +/- 1.9% of initial size; P < 0.05, both comparisons). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect in either system. Prostaglandin E(2) (PGE(2)) inhibited both chemotaxis and gel contraction, and the PDE4 inhibitors shifted the PGE(2) concentration-dependence curve to the left in both systems. The inhibition of endogenous PGE(2) production by indomethacin diminished the effects of the PDE4 inhibitors in both chemotaxis and gel contraction, consistent with the concept that the PDE4 inhibitory effects on fibroblasts are related to the presence of cyclic adenosine monophosphate in the cells. In summary, these in vitro results suggest that PDE4 inhibitors may be able to suppress fibroblast activity and, thus, have the potential to block the development of progressive fibrosis.
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PMID:PDE4 inhibitors attenuate fibroblast chemotaxis and contraction of native collagen gels. 1203 68

Tadalafil is an inhibitor of phosphodiesterase type 5, and is currently undergoing regulatory review in the US and in Europe. Its chemical structure is significantly different from sildenafil, and in vitro studies confirm significant potency for PDE5 inhibition, with little activity against most of the other isoforms of the enzyme including PDE6, which is the isoform of the enzyme found within the retina. The half-life of tadalafil is 17.5 hours and clinical studies suggest significant activity 24 hours post-dosing. As with sildenafil, efficacy depends upon a normal sexual stimulus, and the drug can taken be as required. Tadalafil is effective in the treatment of men with erectile dysfunction, and it appears to have a relatively mild side-effect profile, with no visual side-effects noted.
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PMID:Tadalafil (Cialis) for men with erectile dysfunction. 1207

The effects of cGMP binding on the catalytic activity of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) are unclear because cGMP interacts with both allosteric and catalytic sites specifically. We studied the effects of cGMP on the hydrolysis of a fluorescent substrate analogue, 2'-O-anthraniloyl cGMP, by PDE5 partially purified from rat cerebella. The preparation contained PDE5 as the major cGMP-PDE activity and was not contaminated with cAMP- or cGMP-dependent protein kinases. The Hill coefficients for hydrolysis of the analogue substrate were around 1.0 in the presence of cGMP at concentrations <0.3 microM, while they increased to 1.5 at cGMP concentrations >1 microM, suggesting allosteric activation by cGMP at concentrations close to the bulk binding constant of the enzyme. Consistent with an allosteric activation, increasing concentrations of cGMP enhanced the hydrolysis rate of fixed concentrations of 2'-O-anthraniloyl cGMP, which overcame competition between the two substrates. Such activation was not observed with cAMP, cyclic inosine 3',5'-monophosphate, or 2'-O-monobutyl cGMP, indicating specificity of cGMP. These results demonstrate that cGMP is a specific and allosteric activator of PDE5, and suggest that in cells containing PDE5, such as cerebellar Purkinje cells, intracellular cGMP concentrations may be regulated autonomously through effects of cGMP on PDE5.
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PMID:Allosteric activation of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) by cGMP. 1213 89

Sildenafil is a potent and selective inhibitor of the cyclic GMP-specific phosphodiesterase (PDE5) that is very effective in the treatment of male impotence. It inhibits breakdown of cyclic guanosine monophosphate (cGMP) formed in penile smooth muscle cells in response to stimulation by nitric oxide resulting in muscle relaxation. PDE5 is widely distributed in the body, being present in the vasculature, platelets, and kidneys. In the kidney, PDE5 is involved in the regulation of sodium excretion and renin secretion. The aim of the present investigation was to investigate the effect of sildenafil, in doses used clinically, on renin secretion in human subjects. The studies were performed in two groups of healthy normotensive subjects: one in which sodium intake was unrestricted, and one in which sodium intake was restricted to 600 mg/day. Blood pressure and heart rate were monitored throughout the study, and blood samples for the measurement of plasma cGMP and cAMP concentrations and plasma renin activity (PRA) were collected. After control measurements, the subjects ingested a capsule containing sildenafil or placebo. Cardiovascular measurements and blood sampling continued for the next 120 min. Sildenafil had only minor cardiovascular effects. Diastolic pressure tended to be lower and heart rate was generally higher after sildenafil than after placebo, but the differences were small. Sildenafil caused a prompt and sustained increase in plasma cGMP concentration and a more gradual increase in plasma cAMP concentration. After the subjects received placebo, there was a progressive decrease in PRA during the 2-hr observation period, presumably reflecting the circadian rhythm in renin secretion. In contrast, PRA failed to decrease after the subjects received sildenafil, thus indicating that sildenafil exerts a stimulatory action on renin secretion. This action on renin secretion may help explain why sildenafil only has minor effect on blood pressure despite the widespread distribution of PDE5 in vascular tissues.
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PMID:Effect of sildenafil on renin secretion in human subjects. 1219 4

Developmental changes (from 2 to 26 weeks) in phosphodiesterase (PDE) activity in the rat submandibular gland were investigated. Major activities for both cAMP- and cGMP-PDE were present in the 100000 x g supernatant fractions (70-90% of total activities), but not in the pellet fractions, during development. The effects of stimulators (Ca(2+)/calmodulin and cGMP) and inhibitors (cGMP, cilostamide, rolipram and zaprinast) were investigated in the supernatant fractions. During development, PDE4 (cAMP-specific PDE) was a major PDE, indicating that the majority of cAMP is hydrolysed by PDE4. In the young rat, PDE1 hydrolysed cGMP three-fold more than the control, and PDE2 (cGMP-stimulated PDE) was present, indicating that the concentration of intracellular cGMP may be enhanced, and cGMP may function in the growth pathway in the submandibular gland. Chromatograms eluted on a Mono Q HR5/5 ion-exchange column supported the results of the inhibition studies: PDE1, PDE2, PDE3, PDE4 and PDE5 were present in the young submandibular gland, and PDE1, PDE3, PDE4 and PDE5 in the adult gland. Expression of PDE5 was detected by inhibition studies, reverse transcriptase-polymerase chain reaction and Western blotting in the submandibular gland.
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PMID:Changes in phosphodiesterase activity in the developing rat submandibular gland. 1222 Oct 13

Tissue destruction, resulting in emphysema, can be a consequence of several pathologic processes. The current study evaluated the effects of the phosphodiesterase (PDE)4 inhibitor, cilomilast, and other PDE inhibitors on the ability of fibroblasts to degrade extracellular matrix. Using the three-dimensional collagen gel culture system, fibroblasts (HFL-1) were cultured with tumor necrosis factor (TNF)-alpha, known to induce matrix metalloproteinase (MMP) release, and/or neutrophil elastase (NE), which can induce MMP activation. On Day 4, gels containing TNF-alpha and NE were significantly degraded (20.8 +/- 2.9% of original collagen content). Cilomilast (10 micro M) inhibited this degradation (84.4 +/- 8.4%). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect. Gelatin zymography and immunoblotting revealed that fibroblasts cultured with TNF-alpha released increased amounts of latent MMP-1 and -9. The addition of NE resulted in the conversion of MMP-1 and -9 to their active forms, indicative of collagen degradation. Cilomilast inhibited the release of MMP-1 and -9, as well as conversion of MMP-1 to its active form. Using real-time PCR analysis, cilomilast's effect on MMP-1 release was not associated with the proteinase's mRNA expression, suggesting that the inhibition of release is regulated at the post-transcriptional level. These results suggest that cilomilast may be a potentially effective therapeutic agent in diseases characterized by excessive tissue destruction, such as emphysema.
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PMID:Phosphodiesterase 4 inhibitor cilomilast inhibits fibroblast-mediated collagen gel degradation induced by tumor necrosis factor-alpha and neutrophil elastase. 1235 83

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.
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PMID:Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity. 1235 32

Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3'5' cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-alpha (SDF-1alpha). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.
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PMID:Sildenafil and vardenafil, types 5 and 6 phosphodiesterase inhibitors, induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia cells. 1239 51


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