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)

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.
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PMID:Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins. 629 46

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.
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PMID:Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurospora crassa. 630 27

Cyclic nucleotide phosphodiesterase (0.07 nM) was activated by near stoichiometric concentrations of [3-(2-pyridyldithio)propionyl]calmodulin (PDP-CaM) after initial incubation of these proteins at 200-fold higher concentrations; activity in assays with EGTA was 80% of that in the presence of Ca2+. The enzyme incubated with native calmodulin under identical conditions required approximately 1 nM for half-maximal activation, and no activation was observed in the absence of calcium. These data suggested formation of a covalent complex between phosphodiesterase and PDP-CaM. On high-performance gel-permeation chromatography in the presence of metal chelators, the complex appeared considerably larger than the native enzyme. Incubation of phosphodiesterase with the thiolated (inactivated) form of PDP-CaM did not change its chromatographic behavior, indicating that reactive sulfhydryl groups were involved in complex formation. Although the total activities recovered from chromatography were not significantly different, maximal activation of PDP-CaM-phosphodiesterase complex was only approximately 20%, whereas the control enzyme was activated 6-8-fold by Ca2+ plus calmodulin. Kinetics of cGMP hydrolysis in the presence of EGTA by the isolated complex differed from those of control enzyme but were indistinguishable from those of control enzyme assayed with saturating Ca2+ and CaM. The calmodulin antagonists W-7 and trifluoperazine had relatively little effect on activity of the PDP-CaM-phosphodiesterase complex. Incubation of the complex with dithiothreitol dramatically increased its Ca2+ and calmodulin responsiveness, suggesting that reduction of the disulfide cross-link released phosphodiesterase from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation of an enzymatically active cross-linked complex between brain cyclic nucleotide phosphodiesterase and 3-(2-pyridyldithio)propionyl-substituted calmodulin. 632 62

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.
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PMID:Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue. 633 97

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

n-3 and n-6 polyunsaturated fatty acids are involved in the regulation of the immune response. Although different hypotheses related to modifications of arachidonic acid metabolism or alterations at the level of the cell membrane have been put forward to explain their suppressive effect on the lymphocyte growth, their mechanism of action remains largely unknown. Cyclic nucleotide phosphodiesterase (PDE) has been shown to be an important target involved in the control of lymphocyte proliferation. The present study aimed to determine whether in vitro addition of a physiological concentration (5 microM) of n-6 (20:3n-6) or n-3 (18:4n-3, 20:5n-3, 22:6n-3) fatty acids to human peripheral blood mononuclear cells (PBMC) was able to alter the PDE activity of these cells, and especially the PDE increase in response to Con A stimulation. Pretreatment of human PBMC for a short period of time (90 min) with 5 microM of either 20:3n-6, 20:5n-3 or 22:6n-3 was sufficient to induce a significant enrichment of cellular phospholipids in the corresponding fatty acid, whereas 18:4n-3 was poorly incorporated. Either fatty acid significantly increased both cAMP- and cGMP-PDE activities in the cytosolic compartment, the particulate PDE activities being less sensitive to their stimulatory effect. In contrast, they significantly lowered the PDE increase to Con A stimulation. Except 20:5 n-3, the three other fatty acids did not alter significantly the basal or Con A-induced oxygenated metabolism of arachidonic acid (AA), appreciated by the measurement of radioactive eicosanoids formed in [3H]AA-labelled cells. Furthermore, only 20:5n-3 significantly inhibited the lymphoproliferative response to Con A, whereas 16:0, 18:0, 18:1n-9, 20:3n-6 and 20:4n-6 were inactive. The inhibitory effect was not prevented by antioxidant vitamins C and E. The present results suggest that the lymphocyte growth suppressive effect of 20:5n-3 20:5n-3 is very likely to be independent on both the cAMP system and eicosanoid synthesis, and does not seem to involve their conversion to peroxidised products.
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PMID:Influence of polyunsaturated fatty acids on lipid metabolism in human blood mononuclear cells and early biochemical events associated with lymphocyte activation. 772 18

Cyclic nucleotide phosphodiesterase (PDE) isoenzymes (I-V) have been demonstrated in airways smooth muscle of several species including man. Theophylline is a non-selective inhibitor of PDE and is a potent relaxant of airways smooth muscle but its use is limited by its toxicity. Consequently, research into new, isoenzyme-selective PDE inhibitors is seen as important. The potential airways smooth muscle relaxant effects of these drugs is discussed in this review. Cyclic AMP PDE (types III and IV) inhibition produces greater relaxation than cyclic GMP PDE (types I and V) inhibition. No PDE II-selective inhibitors have been described. Airways smooth muscle relaxation in vitro, is greater with PDE IV than PDE III inhibitors in guinea-pig and bovine airways whereas PDE III is more important in porcine airways. Both cyclic AMP PDEs are important in human airways. PDE III or IV inhibition can produce additive effects and can augment isoprenaline actions. PDE V inhibition augments sodium nitroprusside-induced effects. There are no reported interactions between cyclic AMP and cyclic GMP PDE inhibitors. In vivo, cyclic AMP PDE inhibitors are more potent bronchodilators than cyclic GMP PDE inhibitors. PDE IV inhibitors have less cardiovascular side-effects. Topical administration may further increase efficacy and selectivity. Clinically PDE III inhibition improves lung function but also affects cardiovascular parameters. Inhaled PDE III/IV inhibitors produce bronchodilation without marked side effects. Potent, selective PDE IV inhibitors are currently being evaluated. In conclusion, isoenzyme-selective PDE inhibitors, especially PDE IV, may be useful airways smooth muscle relaxants in the treatment of lung disorders such as asthma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isoenzyme-selective cyclic nucleotide phosphodiesterase inhibitors: effects on airways smooth muscle. 775 80

An increase in cyclic nucleotide monophosphate levels is suggested to play a prominent role in mediating smooth-muscle relaxation. Cyclic nucleotide phosphodiesterase (PDE) influences smooth-muscle tone by decreasing the level of cyclic nucleotides. At present, five different families of isoenzymes of PDE exist that show a distinct species- and organ-specific distribution. Our study was done to evaluate the existence of specific PDE isoenzymes and its functional role in human ureteral tissue. Normal ureteral tissue was homogenized and centrifuged and the supernatant fraction was separated using anioin-exchange diethylaminoethyl (DEAE)-Sephacel chromatography. A PDE assay was then performed and the peak fractions were added to different specific PDE activators and inhibitors. In vitro, longitudinal ureteral strips were precontracted and different selective and non-selective PDE inhibitors were added incremently. Three different PDE isoenzymes were characterized: PDE I (calmodulin-sensitive), PDE II (cGMP-stimulated), and PDE IV (cAMP-specific). All PDE inhibitors relaxed the strips dose-dependently, with the 50% effective concentrations (EC50) being 30 microM for papaverine, 40 microM for zaprinast, 25 microM for quazinone, and 0.1 microM for rolipram. The ureter-relaxing effect of the PDE IV inhibitor at low concentrations, combined with its low-level effect on the systemic circulatory parameters, may open the possibility of using selective PDE IV-inhibitors in the treatment of ureteral colics or for ureteral stone passage.
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PMID:Characterization of cyclic nucleotide phosphodiesterase isoenzymes in the human ureter and their functional role in vitro. 786 26

To investigate a possible physiological desensitization process for beta 3-adrenergic responses, the effect of cold acclimation of hamsters on adrenergically stimulated oxygen consumption of isolated brown fat cells was investigated. Cells were prepared from control and from cold-acclimated hamsters. In agreement with earlier findings, cells isolated from cold-acclimated hamsters responded to norepinephrine addition with a decreased sensitivity (approximately 10 times higher 50% effective concentration) and a decreased maximal rate of oxygen consumption compared with cells from control hamsters. When cells were stimulated with the general beta-adrenergic agonist isoprenaline or with the beta 3-selective agonists BRL-37344 or CGP-12177, a similarly desensitized response was observed, demonstrating that it was indeed a beta 3-adrenergic response that was functionally desensitized. However, when the mitochondria within the cells were directly stimulated with exogenous free fatty acids (palmitate or octanoate), no difference between cells from control and cold-acclimated animals was seen, indicating that a mediatory step must be desensitized. When the cells were stimulated with forskolin (to activate adenylyl cyclase) or with 8-bromoadenosine 3',5'-cyclic monophosphate, the desensitized response was still observed. At post-adenosine 3',5'-cyclic monophosphate levels, a desensitization was not evident. Cyclic nucleotide phosphodiesterase activity was increased in cells from cold-acclimated animals. It is therefore suggested that this increased activity of phosphodiesterase could be (at least partly) responsible for the physiologically induced desensitized responses observed here.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Physiological desensitization of beta 3-adrenergic responses in brown fat cells: involvement of a postreceptor process. 790 9

Cyclic nucleotide phosphodiesterase (PDE) enzymes are felt to play a role in the regulation of inflammatory responses through their effects on cAMP. In this study, we investigated the effects of nonselective and isozyme selective PDE inhibitors on the proliferative responses of peripheral blood mononuclear cells (PBMCs) to ragweed (RW, a Th2 stimulus), tetanus toxoid (TT, a Th1 stimulus), and phytohemagglutinin in ragweed-allergic patients. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) produced nearly identical concentration-dependent inhibition for PBMCs cultured with either Ag (RW and TT) or mitogen (phytohemagglutinin). Neither the type V inhibitor, zaprinast, nor the type III inhibitor, siguazodan, was effective at inhibiting antigen- or mitogen-driven proliferative responses of human PBMCs. The type IV inhibitor, rolipram, was unique in its ability to inhibit the RW-driven proliferative response and, to a lesser extent, the TT-driven proliferative response. However, rolipram was ineffective at inhibiting the mitogen-driven proliferative response. Only with a combination of type III and type IV inhibitors could the efficacy on the TT response be made to approximate that of the type IV inhibitor alone in the RW-driven system. Although the efficacies of IBMX and rolipram were identical in the RW-driven system, the IC50 value of the latter was 10-fold lower, similar to the difference noted in the TT-driven system between IBMX alone and the combination of rolipram with siguazodan. Dose-response curves generated by using the D- or L-isomers of rolipram were not appreciably different from each other or from the curve generated with racemic rolipram. The addition of supraphysiologic doses of human rIL-2 and rIL-4 was unable to counteract the inhibitory effect of IBMX or rolipram. These data support the hypothesis that modulation of proliferation of PBMCs by selective PDE inhibitors varies in sensitivity with the type of stimulus used, and that the type IV PDE exerts the predominant cAMP-associated regulatory effect on allergen-driven proliferation. Finally, the inhibitory effect induced by rolipram is independent of its stereochemistry and cannot be exclusively attributed to deficits in IL-2 or IL-4.
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PMID:Modulation of antigen- and mitogen-induced proliferative responses of peripheral blood mononuclear cells by nonselective and isozyme selective cyclic nucleotide phosphodiesterase inhibitors. 793 May 66


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