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)

The cAMP phosphodiesterase (PDE) activity of CHO cells was unaffected by the addition of Ca2+ +calmodulin (CaM), indicating the absence of any PDE1 (Ca2+/CaM-stimulated PDE) activity. Treatment with the tumour promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) led to the rapid transient induction of PDE1 activity which attained a maximum value after about 13 h before slowly decreasing. Such induction was attenuated by actinomycin D. PCR primers were designed to hybridize with two regions identified as being characteristic of PDE1 forms found in various species and predicted to amplify a 601 bp fragment. RT-PCR using degenerate primers allowed an approx. 600 bp fragment to be amplified from RNA preparations of rat brain but not from CHO cells unless they had been treated with PMA. CHO cells transfected to overexpress protein kinase C (PKC)-alpha and PKC-epsilon, but not those transfected to overexpress PKC-beta I or PKC-gamma, exhibited a twofold higher PDE activity. They also expressed a PDE1 activity, with Ca2+/CaM effecting a 1.8-2.8-fold increase in total PDE activity. RT-PCR, with PDE1-specific primers, identified an approx. 600 bp product in CHO cells transfected to overexpress PKC-alpha and PKC-epsilon, but not in those overexpressing PKC-beta I or PKC-gamma. Treatment of PKC-alpha transfected cells with PMA caused a rapid, albeit transient, increase in PDE1 activity, which reached a maximum some 1 h after PMA challenge, before returning to resting levels some 2 h later. The residual isobutylmethylxanthine (IBMX)-insensitive PDE activity was dramatically reduced (approx. 4-fold) in the PKC-gamma transfectants, suggesting that the activity of the cyclic AMP-specific IBMX-insensitive PDE7 activity was selectively reduced by overexpression of this particular PKC isoform. These data identify a novel point of 'cross-talk' between the lipid and cyclic AMP signalling systems where the action of specific PKC isoforms is shown to cause the induction of Ca2+/CaM-stimulated PDE (PDE1) activity. It is suggested that this protein kinase C-mediated process might involve regulation of PDE1 gene expression by the AP-1 (fos/jun) system.
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PMID:Induction of Ca2+/calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary cells (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms. 757 35

1. CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2. The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7-like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3. By cDNA-PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4. No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5. Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time- and concentration-dependent manner, which was increased in the presence of interleukin-4 (IL-4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Sp-isomer (dcl-cBIMPS), respectively. However, at concentrations exceeding 100 microM db-cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 microM) and forskolin (10 microM) did not affect B cell proliferation, even when given in combination with rolipram. 6. Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp-8-Br-cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7. Importantly, PDE4 activity in LPS/IL-4-activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8. We conclude that an increase in cyclic AMP, mediated by down-regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL-4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.
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PMID:Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation. 955 83

Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.
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PMID:Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells. 992 21

The inhibitory potential of novel anti-platelet aggregatory cilostamide analogues on phosphodiesterase (PDE) isozyme activities was investigated with recombinant PDE isozymes expressed in a baculovirus/ Sf9 expression system. The recombinant enzymes (PDE1-PDE5 and PDE7) showed Km values and sensitivities to selective inhibitors similar to those reported previously for native enzymes purified from tissues. The cyclooctylurea derivative OPC-33540 (6-[3-[3-cyclooctyl-3-[(1R*,2R*)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone) inhibited recombinant PDE3A (IC50 = 0.32 nM) more potently and selectively than the classical PDE3 inhibitors cilostamide, cilostazol, milrinone, and amrinone. The cyclopropylurea derivative OPC-33509 [(-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone] was less potent (IC50 = 0.10 microM) than OPC-33540, demonstrating that the cyclooctyl moiety was important for a potent inhibitory effect. In platelets, OPC-33540 potentiated cyclic AMP accumulation concentration-dependently in both the absence and the presence of 3 nM prostaglandin E1 (PGE1) (doubling concentrations: 32.5 and 6.2 nM, respectively). OPC-33540 inhibited thrombin-induced platelet aggregation potently (Ic50 = 27.8 nM). The anti-platelet aggregation effect also was stimulated in the presence of 3 nM PGE1 (IC50 = 6.0 nM). There was a good correlation between the IC50 values of PDE3 inhibitors in this study for recombinant PDE3A activity and their IC50 values for thrombin-induced platelet aggregation (r = 0.998). These data demonstrated that OPC-33540 is a highly selective and potent PDE3 inhibitor and a useful probe for identification of the intracellular functions of PDE3.
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PMID:Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity. 1064 42

Certain subsets of lymphoid cells, such as thymocytes or peripheral B cells, undergo apoptosis after treatment with agents which elevate intracellular 3',5' cyclic adenosine monophosphate (cAMP). Investigators have also noted induction of apoptosis of chronic lymphocytic leukemia (CLL) cells following treatment with methylxanthines, a phenomenon that may, at least in part, be due to the activity of these drugs as non-specific phosphodiesterase (PDE) inhibitors. We discuss three general strategies for altering cAMP-mediated signal transduction in lymphoid cells. After a review of what is known about the expression and regulation of PDE families in human lymphoid cells, we focus on the use of isoform-specific PDE inhibitors as potential therapeutic agents in CLL. Our work has suggested that despite the presence of PDE1, PDE3B, PDE4 and PDE7 enzymes in CLL, inhibition of PDE4 results in uniquely potent induction of apoptosis in CLL cells. This effect is relatively specific as comparable treatment of human peripheral blood T cells does not induce apoptosis. Clinical trials utilizing PDE4 inhibitors are indicated in the therapy of CLL patients resistant to standard therapy.
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PMID:The cAMP signaling pathway as a therapeutic target in lymphoid malignancies. 1072 68

To identify amino acids that might be involved in discriminating guanosine-3',5'-cyclic phosphate (cGMP) towards adenosine-3',5'-cyclic phosphate (cAMP) binding in the cAMP-specific phosphodiesterases, alignments of different human cyclic nucleotide phosphodiesterases (PDEs) were performed. Eight amino acid residues that are highly conserved in the cAMP-hydrolysing phosphodiesterases (PDE1, PDE3, PDE4, PDE7, PDE8) and that did not show any homologies to the cGMP-specific phosphodiesterases (PDE5, PDE6, PDE9) were selected from these alignments. Using the technique of site-directed mutagenesis, derivatives of PDE4A carrying single mutations at these conserved residues (amino acid positions are given according to the human PDE4A isoform HSPDE4A4B; accession number L20965) were generated and expressed in COS1 cells. The expression products were characterised with regard to cAMP and cGMP hydrolysis and sensitivity towards type-specific inhibitors. The mutation of Phe484 toward Tyr, Ala590 toward Cys, Leu391 and Val501 towards Ala had no significant influence on substrate affinity or specificity. However, the exchange of Trp375 and Trp605 for aliphatic residues abolished catalytic activity and the exchange of Pro595 for Ile led to sevenfold decrease of substrate affinity and an 14-fold decrease of the affinity towards the PDE4-specific inhibitor 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram). Both effects may provide evidence for a structural importance of Trp375, Trp605 and Pro595 for PDE function. By exchanging the aspartate residue for asparagine or alanine at position 440 of the human PDE4A4B isoform, the substrate specificity was altered from the highly specific cAMP hydrolysis to an equally efficient cAMP and cGMP binding and hydrolysis. In addition, the IC(50) values for common PDE4-specific inhibitors like rolipram, N-(3,5-dichlorpyrid-4-yl)-3-cyclopentyl-oxy-4-methoxy-benzamide (RPR-73401) and 8-methoxy-5-N-propyl-3-methyl-1-ethyl-imidazo[1,5-a]-pyrido[3,2-e]-pyrazinone (D-22888) were dramatically increased. These results demonstrate an important role of the aspartate at position 440 in determining substrate specificity and inhibitor susceptibility of PDE4A. The strong conservation of this residue suggests that Asp440 may play a similar role in other cAMP-PDEs.
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PMID:Identification of substrate specificity determinants in human cAMP-specific phosphodiesterase 4A by single-point mutagenesis. 1128 54

We studied the activities of 3',5'-adenosine-cyclic monophosphate (cAMP)- synthesizing adenylate cyclase (AC) and cAMP-hydrolyzing cyclic nucleotide phosphodiesterase (PDE) in phytohemagglutinin (PHA)- or anti-CD3 plus anti-CD28-stimulated human T cells, and examined their roles in interleukin-13 (IL-13) production. The AC inhibitor MDL 12,330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride] completely blocked PHA- or anti-CD3/CD28-induced IL-13 production. The PDE 1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine or the PDE4 inhibitor rolipram partially inhibited IL-13 production, and the addition of both resulted in 100 or 85% inhibition in PHA- or anti-CD3/CD28-stimulated T cells, respectively. AC in T cells was transiently activated 5 min after stimuli, followed by the transient activation of PDE4 at 30 min. PDE1 activity, undetectable in resting T cells, was detected 3 hr after stimuli, and then increased gradually. Although PDE1-, 2-, 3-, and 4-independent PDE activity was low (< or = 15% of total), it began to increase 3 hr after anti-CD3/CD28; the increase was blocked by PDE7 antisense oligonucleotide, and such an increase was not induced by PHA. PHA or anti-CD3/CD28 induced PDE1B mRNA expression, undetectable in resting T cells. PDE4 mRNA level in T cells was not altered by either stimulus. PDE7 mRNA expression was detected in resting T cells, and was enhanced by anti-CD3/CD28, but not by PHA. The cAMP level of T cells increased 5 min after stimuli, returned to the basal level at 2 hr, and then continued to decrease. These results suggest that PHA or anti-CD3/CD28 initially (< or = 5 min) increases cAMP in T cells via AC, then reverses the increase via PDE4 (< or = 2 hr), and in the later phase (> 2 hr) further decreases cAMP via PDE1. Both the time-dependent increase and decrease of cAMP may be required for IL-13 production.
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PMID:Regulatory roles of adenylate cyclase and cyclic nucleotide phosphodiesterases 1 and 4 in interleukin-13 production by activated human T cells. 1144 60

The cyclic monophosphate nucleotides (cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]) are found ubiquitously in mammalian cells and act as second messenger transducers to effect the intracellular actions of a variety of hormones, cytokines, and neurotransmitters. In turn, these nucleotides also modulate the signal transduction processes regulated by a range of cytokines and growth factors. Previously, we have reported that pentoxifylline, a nonselective phosphodiesterase (PDE) inhibitor, can promote osteoblastic differentiation by elevating intracellular cAMP levels and, consequently, enhance bone formation in vivo and in vitro. In this study, reverse-transcription polymerase chain reaction (RT-PCR) analysis of the osteoblastic cell lines, MC3T3-E1 and ST2 revealed the presence of PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, and PDE9. We examined the effect of selective inhibitors for a respective PDE isozyme on the capacity of bone morphogenetic protein 4 (BMP-4)-induced alkaline phosphatase (ALP) activity, a cellular differentiation marker, in cells with osteogenetic potential. The results indicate that selective inhibitors for PDE2, PDE3, and PDE4 enhanced the BMP-4-induced ALP activity in a dose-dependent manner in ST2 cells but not in MC3T3-E1 cells. Northern blot analysis also revealed that the selective inhibitors for PDE2, PDE3, and PDE4 enhanced the levels of expression of messenger RNAs (mRNAs) of ALP, osteopontin (OP), and collagen type I in ST2 cells but not in MC3T3-E1 cells except for the treatment with PDE4 inhibitor. Given these data, we conclude that PDE isozymes are involved in the modulation of osteoblastic differentiation mainly at an early stage. Additionally, selective inhibitors for PDE2, PDE3, and PDE4 appear to promote the differentiation of osteogenic precursor cells toward an osteoblastic phenotype.
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PMID:Involvement of phosphodiesterase isozymes in osteoblastic differentiation. 1181 55

PDE7A is a recently described 3',5'-cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) whose expression has been detected in T-cells. As treatment with the methylxanthine theophylline, a nonspecific PDE inhibitor, induces apoptosis in leukemic cells from patients with the B-lineage malignancy chronic lymphocytic leukemia (CLL), we sought to determine if PDE7A was a target of theophylline therapy in such cells. Western analysis revealed expression of PDE7A in normal human splenic B-cells, primary CLL cells, and in a CLL-derived cell line (WSU-CLL). Among the six cAMP PDEs (PDE1B, PDE3B, PDE4A, PDE4B, PDE4D, and PDE7) examined in WSU-CLL, only PDE7A levels were augmented by treatment with methylxanthines. The activity of PDE7A isolated from the WSU-CLL cell line by immunoprecipitation was inhibited by theophylline and IBMX with IC50 values of 343.5 and 8.6 microM, respectively. WSU-CLL PDE7A was also up-regulated by a novel specific inhibitor (IC242), which inhibits PDE7A from WSU-CLL cells with an IC50 value of 0.84 microM. IC242-mediated up-regulation of PDE7A was blocked by the protein kinase A (PKA) inhibitor H-89.
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PMID:PDE7A is expressed in human B-lymphocytes and is up-regulated by elevation of intracellular cAMP. 1181 56

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.
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PMID:Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors. 1206


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