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)

COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE4A5). This led to the expression of a novel, cyclic AMP-specific, rolipram-inhibited phosphodiesterase activity. In such transfected cells a novel approximately 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant. RD1 (RNPDE4A1A). RPDE-6 activity and immunoreactivity was found distributed between both pellet (approximately 25%) and cytosol (approximately 75%) fractions of transfected COS-7 cells. Soluble and pellet RPDE-6 activities exhibited similar low Km values for cyclic AMP (approximately 2.4 microM) and were both inhibited by low concentrations of rolipram, with IC50 values for the soluble activity being lower (approximately 0.16 microM) than for the pellet activity (approximately 1.2 microM). Pellet RPDE-6 was resistant to release by either high NaCl concentrations or the detergent Triton X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an approximately 79 kDa species reflecting RD1 and an approximately 109 kDa species that co-migrated with the immunoreactive species seen in COS cells transfected to express RPDE-6. The approximately 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-transfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immuno-precipitated by these antisera from brain cytosol had the characteristics of COS cell-expressed RPDE-6 with KmcyclicAMP approximately 3.7 microM and IC50rolipram approximately 0.12 microM. The distribution of PDE activity immunoprecipitated from the cytosol of various brain regions paralleled that seen for the distribution of the approximately 109 kDa immunoreactive species. It is suggested that the 109 kDa species identified in brain cytosol and pellet fractions is the native form of RPDE-6. The PDE4A splice variants, RD1 and RPDE-6, were shown to have distinct patterns of expression among various brain regions. PDE4A and PDE4B activities appear to provide the major source of PDE4 activity in brain membranes, whereas the cytosolic PDE4 activity is suggested to reflect predominantly the activity of the PDE4D family. Alternative splicing of the PDE4A gene confers distinct N-terminal domains on RPDE-6 and RD1, which attenuates the Vmax. of these enzymes and defines their distinct subcellular distribution pattern.
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PMID:Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family. 757 34

Cyclic nucleotide phosphodiesterase (PDE) enzymes may participate in regulation of the inflammatory response through their effects on second messengers. In the present study, we have investigated the role of nonselective and isozyme selective PDE inhibitors in altering the antigen-driven cytokine gene expression of peripheral blood mononuclear cells (PBMCs) from atopic individuals. Ragweed and tetanus toxoid were used as model antigens. The nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the selective PDE4 inhibitor, rolipram, markedly suppressed interleukin-5 (IL-5) and interferon gamma (IFN gamma) gene expression in both antigen-driven systems. Gene expression for IL-4 was unaffected by these agents in the ragweed-driven system. Message for IL-4 could not be detected in the tetanus toxoid-driven system, despite the use of a quantitative, competitive reverse transcription-polymerase chain reaction (RT-PCR) assay sensitive to less than 10 fg of target template. The PDE3 inhibitor, siguazodan, was ineffective in downregulating gene expression for the proinflammatory cytokines assayed; when used in combination with the PDE4 inhibitor, the PDE3 inhibitor provided no increase in efficacy over that seen with the PDE4 inhibitor alone. Gene expression for the A and B isoforms of the PDE4 in PBMCs was unaffected by antigen stimulation or treatment with the PDE4 inhibitor; however, differences in expression of these two isoforms were apparent when a variety of immune cell lines were studied. These data support the hypothesis that the primary anti-inflammatory target for PDE inhibition in PBMCs is the PDE4. Furthermore, the expression of various isoforms of this enzyme may differ between immune cell types. Finally, PDE4 isoform expression in PBMCs is independent of treatment with an isozyme selective inhibitor.
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PMID:Effects of nonselective and isozyme selective cyclic nucleotide phosphodiesterase inhibitors on antigen-induced cytokine gene expression in peripheral blood mononuclear cells. 757 7

Many functions of the immune and inflammatory responses are inhibited by agents that increase intracellular levels of cAMP. Recent investigations have revealed that cAMP levels in inflammatory cells are regulated by cyclic nucleotide phosphodiesterases (PDEs) belonging to the PDE4 family (cAMP-specific PDEs). At least four different genes are known to encode PDE4 isozymes, which are characterized by their selectivity for cAMP over cGMP and their sensitivity to the antidepressant drug rolipram. The aim of our studies was to investigate whether monocytic cells could regulate PDE4 activity and whether certain PDE4 isozymes were expressed preferentially over others. Our results showed that treatment of peripheral blood monocytes or closely related Mono Mac 6 cells with dibutyryl-cAMP or other cAMP-elevating agents transiently increased rolipram-sensitive PDE4 activity 2-3-fold, without concomitant increases in cGMP-inhibited PDE (PDE3) activity. PDE4 activity was predominantly cytosolic, whereas PDE3 activity was localized to the particulate fraction. Our Northern and Western blot studies with reagents recognizing three distinct PDE4 gene products (PDE4A, PDE4B, and PDE4D) revealed that their expression is transcriptionally regulated in monocytic cells. Although none of the three isozymes was detectable under normal culture conditions, all of these were up-regulated when Mono Mac 6 cells were exposed to dibutyryl-cAMP. Distinct differences were observed in their temporal patterns of expression. Endotoxin lipopolysaccharide, a potent monocyte stimulus, also enhanced PDE4 activity in monocytic cells. These data indicate that monocytic cells may express different PDE4 isozymes, depending on their state of activation or differentiation. These isozymes could thus regulate intracellular cAMP levels at various stages of monocyte activation and could thereby be important in limiting the inflammatory response.
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PMID:Regulation of distinct cyclic AMP-specific phosphodiesterase (phosphodiesterase type 4) isozymes in human monocytic cells. 760 56

To define essential interactions of cAMP with the catalytic sites of cyclic nucleotide phosphodiesterases (PDEs) and to begin to map the topology of the sites, we have tested a series of cAMP analogs as competitive inhibitors of the PDEs that hydrolyze cAMP with high efficiency (PDE1, PDE2, PDE3, and PDE4). Comparisons of IC50 values, relative to cAMP, were used to predict which functional groups on cAMP interact with each isozyme. Common to all PDEs tested, except for the calcium/calmodulin-dependent PDE (CaM-PDE, PDE1), is an interaction at the N1-position of cAMP and a distinct lack of binding to the 2'-hydroxyl group of the ribose moiety. Only the cGMP-stimulated (PDE2) and cAMP-specific (PDE4) PDEs appear to interact strongly at the N7-position. The cGMP-inhibited PDE (cGI-PDE, PDE3) may interact less strongly with this nitrogen. The PDE4 and PDE3 both interact with cAMP through the 6-amino group, which most likely serves as a hydrogen bond donor. PDE4 and PDE3 appear to be able to bind to the anti-conformer of cAMP, whereas the PDE1 and PDE2 bind the syn-conformer. The CaM-PDE exhibits no appreciable specificity for any of the analogs tested, showing little or no interaction with the 6-amino group or with any of the ring nitrogens. Large differences exist in the nucleotide-binding requirements for the PDE catalytic sites, compared with the regulatory sites of cAMP-dependent protein kinase and the catabolite activator protein.
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PMID:Characterization of cyclic nucleotide phosphodiesterases with cyclic AMP analogs: topology of the catalytic sites and comparison with other cyclic AMP-binding proteins. 787 42

In order to characterize the structure and regulation of members of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel member of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at its 5' end, consistent with alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted protein of 763 amino acids, of which all but 21, located at the extreme amino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 in COS cells produced a protein of 98 +/- 1.4 kDa, as determined by immunoblotting with an antiserum specific to the carboxyl-terminal regions of all PDE4A proteins, compared to a predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39 mRNA only in testis. Immunoblotting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cells expressing pRPDE39. COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activity) and a cytosolic fraction. The particulate fraction had a Km for cAMP of 3.3 +/- 0.6 microM, and the cytosolic fraction a Km of 5.4 +/- 2.8 microM. The Vmax values for the pRPDE39 protein, relative to the RD1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate and cytosolic forms, respectively. The pRPDE39-encoded PDE activity could not be removed from the particulate fraction by high salt concentrations, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibited by rolipram at an IC50 of 0.5 +/- 0.2 microM for the particulate form and 1.0 +/- 0.2 microM for the cytosolic form, which are values typical of PDE4 family members. The highly tissue-specific distribution of the pRPDE39 mRNA suggest that the pRPDE39 protein functions to modulate a cAMP signaling pathway that is present largely, if not exclusively, in the testis.
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PMID:Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8. 855 32

1. Previous studies have provided evidence that activation of beta-adrenoceptors on cholinergic nerve terminals can inhibit neurotransmission in the airways. However, in most cases, this conclusion has been based on indirect evidence obtained from mechanical experiments where changes in airways smooth muscle tone were measured. 2. We have assessed whether modulation of cholinergic neurotransmission by beta-adrenoceptor agonists is due to a pre- or post-junctional action by investigating the effect of isoprenaline on contractile responses evoked by exogenous acetylcholine (ACh) and electrical field stimulation (EFS; 4 Hz, 40 V, 0.5 ms pulse width every 15 s), and on EFS-induced ACh release from cholinergic nerves innervating guinea-pig and human trachea. Furthermore, the subtype of beta-adrenoceptor which modulates neurotransmission and the potential role of cyclic AMP in this response were evaluated. 3. In guinea-pig trachea, isoprenaline (1 nM-1 microM) inhibited the contractile response evoked by exogenous ACh (1 microM) to a similar extent to that evoked by EFS (EC50 = 19.9 and 23 nM, respectively). 4. In epithelium-denuded guinea-pig strips treated with indomethacin (10 microM), isoprenaline significantly enhanced EFS-induced ACh release from cholinergic nerve terminals (by 36% at 0.3 microM). This effect was blocked by propranolol and ICI 118, 551 (each 0.1 microM). In contrast, isoprenaline failed to affect EFS-induced ACh release from parasympathetic nerves innervating human trachea. 5. To evaluate the role of cyclic AMP in the beta-adrenoceptor-induced facilitation of cholinergic neurotransmission, the effects of various cyclic AMP elevating drugs on ACh release were studied. Forskolin (10 microM) significantly augmented (by 17%) EFS-induced ACh release, an effect which was not reproduced by 1,9-dideoxyforskolin (10 microM) which does not activate adenylyl cyclase. Similarly, the cyclic AMP analogue, 8-bromo-cyclic AMP (1 mM) and cholera toxin (1 microgram ml-1) facilitated ACh output by 22 and 47% respectively, whereas prostaglandin E2 (PGE2, 0.1 nM-1 microM) inhibited this response (by 67% at 1 microM). 6. Zardaverine (10 microM), a dual inhibitor of the phosphodiesterase (PDE)3 and PDE4 isoenzyme families, did not affect EFS-induced ACh release and failed to facilitate the actions of either isoprenaline or PGE2. Similarly, neither SK&F 94120 (10 microM) nor rolipram (10 microM), selective inhibitors of PDE3 and PDE4 respectively, significantly affected the release of ACh in response to EFS. 7. The result of this study suggests that isoprenaline facilitates cholinergic neurotransmission in guinea-pig, but not human, trachea by activation of pre-junctional beta 2-adrenoceptors, an effect that may be mediated via activation of the cyclic AMP/cyclic AMP-dependent protein kinase cascade. Furthermore, the data presented herein illustrate the need to undertake direct measurements of neurotransmitter release when examining the effect of agents purported to act pre-junctionally.
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PMID:Paradoxical facilitation of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by isoprenaline. 873 Jul 33

1. We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP-specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2-induced cyclic AMP accumulation and lipopolysaccharide (LPS)-induced TNF alpha production and TNF alpha mRNA expression. 2. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP-inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT-PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 +/- 0.6 nM, n = 3). (+/-)-Rolipram (IC50: 313 +/- 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R-(-)-rolipram was approximately 3 fold more potent than S-(+)-rolipram against cytosolic PDE4. 4. RP 73401 (IC50: 9.2 +/- 2.1 nM, n = 6) was over 50 fold more potent than (+/-)-rolipram (IC50: 503 +/- 134 nM, n = 6) ) in potentiating PGE2-induced cyclic AMP accumulation. R-(-)-rolipram (IC50: 289 +/- 121 nM, n = 5) was 4.7 fold more potent than its S-(+)-enantiomer (IC50: 1356 +/- 314 nM, n = 5). A strong and highly-significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2-induced monocyte cyclic AMP accumulation and their abilities to displace [3H]-rolipram binding to brain membranes. 5. RP 73401 (IC50: 6.9 +/- 3.3 nM, n = 5) was 71 fold more potent than (+/-)-rolipram (IC50: 490 +/- 260 nM, n = 4) in inhibiting LPS-induced TNF alpha release from monocytes. R-(-)-rolipram (IC50: 397 +/- 178 nM, n = 3) was 5.2-fold more potent than its S-(+)- enantiomer (IC50: 2067 +/- 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNF alpha with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]-rolipram binding (r = 0.65, P < 0.01, n = 13). 6. RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml-1)-induced TNF alpha mRNA. 7. The results demonstrate that RP 73401 is a very potent inhibitor of TNF alpha release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over-production of this pro-inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]-rolipram from its high-affinity binding site, suggesting that the native PDE4 in human monocytes exists predominantly in a 'low-affinity' state.
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PMID:Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNF alpha generation from human monocytes by interacting with a 'low-affinity' phosphodiesterase 4 conformer. 876 90

The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.
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PMID:Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. 881 37

We present the in vitro pharmacology of a novel adenosine 3'-5' -cyclic monophosphate-specific phosphodiesterase (PDE) type 4 inhibitor, CP-80633 ((2'S)5-[3-(2' -exobicyclo[2.2.1]-heptyloxy)4-methoxyphenyl] tetrahydro-2(1H)-primidone), which has shown efficacy in phase II clinical trials for atopic dermatitis. CP-80633 inhibits PDE4 isozymes (human lung IC50 = 1.27 microM) in the absence of effects on PDE1, PDE2, PDE3 and PDE5 isozymes (IC50 > 100 microM). It exhibits no significant selectivity for any single cloned PDE4A, B, C or D isoform. CP-80633 inhibits adenosine 3'-5'-cyclic monophosphate hydrolysis in partially purified human peripheral blood monocyte cytosol (IC50 = 3.52 microM), eosinophil membrane (IC50 = 1.10 microM) and T cell membrane (IC50 = 2.28 microM) preparations. Inhibition of eosinophil PDE4 adenosine 3'-5'-cyclic mono-phosphate hydrolysis by CP-80,633 occurs in a noncompetitive manner. Unlike theophylline, CP-80,633 is inactive against ratrain adenosine (A1,A2) receptors. Consistent with its action as a PDE4 inhibitor in whole cells, CP-80633 potentiates PGE1 dependent increases in adenosine 3'-5'-cyclic monophosphate levels in human U937 cells, and in human eosinophils, monocytes and T cells (EC200 approximately 1.0 microM). Consequently, CP-80633 inhibits many inflammatory cell functions including 1) human eosinophil superoxide anion production (IC50 < 0.6 microM), 2 C5a-(IC50 = 0.40 microM) and LTB4-(IC50 = 0.20 microM) mediated guinea pig peritoneal eosinophil chemotaxis and 3) lipopolysac-charide-induced tumor necrosis factor-alpha release from human monocytes (IC50 = 0.219 microM). These data clearly demonstrate that CP-80633 is a selective inhibitor of PDE4 isozymes, and support its potential use as a therapeutic agent for a number of inflammatory and immune disorders.
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PMID:In vitro pharmacology of the novel phosphodiesterase type 4 inhibitor, CP-80633. 881 23

The regulation of endothelial permeability is poorly understood. An increase in endothelial permeability in the pulmonary microvasculature, however, is critical in noncardiogenic pulmonary edema and other diffuse inflammatory reactions. In the present study thrombin and Escherichia coli hemolysin (HlyA), a membrane-perturbing bacterial exotoxin, were used to alter hydraulic permeability of porcine pulmonary artery and human endothelial cell monolayers. We also investigated the pharmacological approach of adenylyl cyclase activation/phosphodiesterase (PDE) inhibition to block endothelial hyperpermeability. Thrombin (1-5 units/ml) and HlyA (0.5-3 hemolytic units/ml) dose and time dependently (> 15 min) increased endothelial permeability. Forskolin, cholera toxin, and prostaglandin E1, which all stimulate adenylyl cyclase activity, abrogated this effect. One mM dibutyryl cAMP, a cell membrane-permeable cAMP analogue, was similarly active. Endothelial hyperpermeability was also reduced dose dependently by inhibitors of different PDE isoenzymes (motapizone, rolipram, and zardaverine, which block PDE3 and/or PDE4). The effectiveness of PDE inhibitors was increased in the presence of adenylyl cyclase activators. Analysis of cyclic nucleotide hydrolyzing PDE activity in lysates of human umbilical vein endothelial cells showed high activities of PDE isoenzymes 2, 3, and 4. Consistent with the functional data PDE3 and PDE4 were the major cAMP hydrolysis enzymes in intact endothelial cells. We conclude that the hyperpermeability of pulmonary endothelial monolayers, evoked by thrombin or HlyA, can be blocked by the simultaneous activation of adenylyl cyclase and inhibition of PDEs, especially of PDE3 and PDE4. The demonstration of PDE isoenzymes 2-4 in human endothelial cells will help optimize this therapeutic approach.
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PMID:Hyperpermeability of pulmonary endothelial monolayer: protective role of phosphodiesterase isoenzymes 3 and 4. 883 Jan 94


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