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)

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) induced significant superoxide production in human neutrophils within 30 minutes after addition of stimulus and the response was complete within 2 hr. Other agents known to prime neutrophils, including LPS and tumor necrosis factor-alpha, lacked activity under the experimental conditions employed. Using a panel of pharmacologic inhibitors, we sought to compare GM-CSF-induced neutrophil superoxide to that produced by cells exposed to N-formyl methionyl-leucyl-phenylalanine (fMet-Leu-Phe) and phorbol 12-myristate 13-acetate (PMA). Each stimulant displayed a different profile. Rolipram, a peak IV phosphodiesterase inhibitor, specifically inhibited neutrophil activation by GM-CSF and fMet-Leu-Phe, while superoxide production stimulated by PMA was unaffected. Staurosporine, a protein kinase C (PK-C) inhibitor, suppressed superoxide production induced by all three neutrophil stimulants. Cytochalasin B totally inhibited superoxide induced by GM-CSF under conditions that promote the fMet-Leu-Phe-induced response. Cytochalasin B did not markedly affect PMA-induced superoxide. The results are consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, but that differences exist in the initial pathways induced by these neutrophil activators. Superoxide secretion from GM-CSF-treated neutrophils appears to be a direct, delayed response that requires assembly of microfilaments during exposure to the cytokine.
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PMID:Effect of recombinant human granulocyte/macrophage colony-stimulating factor on neutrophil superoxide production. 166 43

Chromatographic analysis of 3',5'-cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the cytosol of human neutrophils shows the predominant presence of PDE IV (cAMP specific) and PDE V (cGMP specific). PDE IV is characterized by (1) cAMP selectivity, (2) a KM for cAMP of 1.2 microM and (3) a typical rank order of IC50-values for PDE inhibitors: 0.13, 0.17, 47 and 9.5 microM for PDE IV selective rolipram, PDE III/IV selective zardaverine, PDE III selective motapizone and unselective 3-isobutyl-1-methylxanthine (IBMX), respectively. Functions of polymorphonuclear leukocytes (PMN) such as N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide release and fMLP/thimerosal elicited leukotriene (LT) biosynthesis are inhibited by these PDE inhibitors with the same rank order and even lower IC50-values. Measurements of changes in cytosolic Cai in Fura-2 loaded PMN demonstrate a transient Cai increase after stimulation with 0.1 microM fMLP and an additional sustained elevation of Cai levels in the presence of thimerosal. PDE inhibitors suppress this sustained phase of Cai release with the same rank order of IC50-values as LT biosynthesis. The correlation between fMLP/thimerosal-induced LT biosynthesis and Cai levels reveal a Cai threshold of 150 nM for arachidonic acid metabolism. cAMP levels in PMN were elevated by PDE inhibitors alone by less than 2-fold. In the presence of fMLP however, cAMP was increased up to 10-fold and the efficacy of PDE inhibitors to increase cAMP paralleled their potency to inhibit PDE IV.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Influence of selective phosphodiesterase inhibitors on human neutrophil functions and levels of cAMP and Cai. 166 89

It is uncertain whether adenosine 3',5'-cyclic monophosphate (cAMP) or the inositol-calcium pathway mediates the stimulation of bone resorption by parathyroid hormone (PTH). Incubation of bone organ cultures with cAMP analogues and forskolin has not resolved this question because of the cellular inhomogeneity of bone and the consequent presence of adenylate cyclase-linked receptors for both PTH and calcitonin, hormones with opposite effects on bone resorption. We have used two new inhibitors of adenylate cyclase, 9-(tetrahydro-2-furyl)adenine (SQ 22536) and 2',5'-dideoxyadenosine (DDA), to directly reassess the role of cAMP in PTH-stimulated osteolysis. SQ 22536 (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blocked PTH stimulation of cAMP production measured in the absence of a phosphodiesterase blocker. In the presence of 1 mM 3-isobutyl-1-methylxanthine, half-maximal inhibition of PTH-induced cAMP production occurred with 0.2 mM SQ and 0.1 mM DDA, respectively. These concentrations of SQ and DDA had no effect on PTH-stimulated 45Ca release from calvaria, although both agents inhibited bone resorption when present at concentrations of 1-2 mM. At these levels, SQ and DDA caused equivalent inhibition of 45Ca release stimulated by 1,25-dihydroxyvitamin D3 but did not affect basal 45Ca release or [3H]-phenylalanine incorporation. It is concluded that substantial blockade of PTH-induced cAMP production does not affect this hormone's stimulation of bone resorption, which is therefore likely to be mediated by another intracellular messenger system, possibly calcium. In millimolar concentrations, SQ and DDA appear to be nonspecific blockers of osteoclastic bone resorption.
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PMID:Adenylate cyclase blockers dissociate PTH-stimulated bone resorption from cAMP production. 169 85

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.
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PMID:A cystic fibrosis pancreatic adenocarcinoma cell line. 169 30

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been characterized in various tissues and cells according to their substrate specificity, intracellular location, and calmodulin dependence. The purpose of this study was to evaluate the possible involvement of different molecular forms of phosphodiesterase in regulating the respiratory burst and lysosomal enzyme release responses of human neutrophils. Treatment with the selective cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase inhibitors Ro 20-1724 or rolipram, or the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), resulted in inhibition of respiratory burst stimulated by the chemoattractants formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (IC50 values: 0.71-17 microM) and complement fragment C5a (IC50 values: 61-93 microM), but did not inhibit phagocytosis-stimulated respiratory burst (less than 10% inhibition at 100 microM). Selective inhibitors of calmodulin-dependent phosphodiesterase (ICI 74,917), calmodulin-insensitive, cyclic GMP-specific phosphodiesterase (M & B 22,948), cyclic GMP-stimulated phosphodiesterase (AR-L 57), or cyclic AMP-specific, cyclic GMP-inhibited phosphodiesterase (amrinone and cilostamide) exhibited little or no inhibitory effect on FMLP- or phagocytosis-stimulated respiratory burst (0-42% inhibition at 100 microM). Regulation of neutrophil activation by phosphodiesterase was also response specific, as Ro 20-1724, rolipram and IBMX were less potent inhibitors of FMLP-induced lysosomal enzyme release (0-14% inhibition at 100 microM). Analysis of human neutrophil preparations confirmed the existence of a cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase, which was associated with the particulate fraction of the cell. These results demonstrate a role for the cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase in the regulation of human neutrophil functions, which appears to be both stimulus specific and response specific.
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PMID:Differential inhibition of human neutrophil functions. Role of cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase. 169 20

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.
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PMID:Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase. 169 84

To investigate the inhibitory effect of adenosine released by endothelium on neutrophil superoxide (O2-) production, we treated confluent monolayers of cultured human umbilical vein endothelial cells with the enzyme adenosine deaminase, and then added human neutrophils. Superoxide (O2-) production by human neutrophils stimulated with 10(-6) M formyl-methionyl-leucyl-phenylalanine was inhibited by 49% in the presence of a confluent monolayer of human umbilical vein endothelial cells (5.1 +/- 0.1 versus 2.6 +/- 0.3 nmols O2-/10(6) neutrophils). Addition of 0.25 U/ml adenosine deaminase to neutrophils plus endothelial cells restored formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil superoxide production to the level seen with neutrophils alone. Deoxycoformycin (10(-4) M), an inhibitor of adenosine deaminase activity, prevented the increase in superoxide production associated with adenosine deaminase addition. The adenosine analogue 5'-(N-ethylcarboxamido)- adenosine (3 x 10(-4) M) caused increased inhibition of formyl-methionyl-leucylphenylalanine-stimulated superoxide release by neutrophils in the presence of endothelial cells and prevented neutrophil-mediated endothelial cell damage, as measured by release of 3H-2-deoxy-D-glucose. Pairing 2-chloroadenosine (10(-5) M) or 5'-(N-ethylcarboxamido)-adenosine (3 x 10(-4) M) with a cyclic adenosine monophosphate phosphodiesterase inhibitor, 3-isobutyl-l-methyl-xanthine (10-4 M), produced greater inhibition of neutrophil superoxide production than occurred with either compound alone. The results support the hypothesis that vascular endothelial cells protect themselves from neutrophil attack by releasing adenosine to inhibit superoxide production.
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PMID:Inhibition of neutrophil superoxide production by adenosine released from vascular endothelial cells. 171 81

The microfilament lattice, composed primarily of filamentous (F)-actin, determines in large part the mechanical (deformability) properties of neutrophils, and thus may regulate the ability of neutrophils to transit a microvascular bed. Circulating factors may stimulate the neutrophil to become rigid and therefore be retained in the capillaries. We hypothesized that cell stiffening might be attenuated by an increase in intracellular cAMP. A combination of cell filtration and cell poking (mechanical indentation) was used to measure cell deformability. Neutrophils pretreated with dibutyryl cAMP (db-cAMP) or the combination of prostaglandin E2 (PGE2, a stimulator of adenylate cyclase) and isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) demonstrated significant inhibition of the n-formyl-methionyl-leucyl-phenylalanine (fMLP)-inducing stiffening. The inhibition of cell stiffening was associated with an increase in intracellular cAMP as measured by enzyme-linked immunoassay (EIA) and an increase in the activity of the cAMP-dependent kinase (A-kinase). Treatment with PGE2 and IBMX also resulted in a decrease in the F-actin content of stimulated neutrophils as assayed by NBD-phallacidin staining and flow cytometry or by changes in right angle light scattering. Direct addition of cAMP to electropermeabilized neutrophils resulted in attenuation of fMLP-induced actin assembly. Neutrophils stimulated with fMLP demonstrated a rapid redistribution of F-actin from a diffuse cortical location to a peripheral ring as assessed by conventional and scanning confocal fluorescence microscopy. Pretreatment of neutrophils with the combination of IBMX and PGE2 resulted in incomplete development and fragmentation of the cortical ring. We conclude that assembly and redistribution of F-actin may be responsible for cell stiffening after exposure to stimulants and that this response was attenuated by agents that increase intracellular cAMP, by altering the amount and spatial organization of the microfilament component of the cytoskeleton.
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PMID:Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP. 171 33

Tibenelast (LY186655), 5,6,-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt, is an orally active anti-anaphylactic compound in guinea pigs, and has been shown to prevent bronchospasm in moderately severe asthmatic patients. Pharmacological studies with tibenelast demonstrated that it is a selective phosphodiesterase (PDE) inhibitor in that it is moderately active against the lung and stomach enzyme while being a very weak inhibitor of the heart enzyme. The compound was without cardiovascular effects at anti-anaphylactic doses. In contrast to theophylline, tibenelast did not have a direct inotropic effect in the cat papillary muscle system. The concentration that inhibited 50% of the enzymatic activity (IC50) for tibenelast was 20- to 30-fold lower for neutrophil PDE than for PDE of other tissues. It was 100 times more potent than aminophylline in inhibiting superoxide generation from platelet-activating factor (PAF)-primed polymorphonuclear leukocytes (PMNL) challenged with chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. However, tibenelast was less effective in the tumor necrosis factor-primed system, and did not inhibit superoxide generation during phagocytosis or when other soluble stimuli, such as phorbo-12-myristate-13-acetate or the calcium ionophore A23187, were used. Furthermore, tibenelast did not inhibit enzymes involved in arachidonic acid metabolism. These results suggest that tibenelast probably inhibits superoxide release from PMNL via a selective inhibition on PDE.
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PMID:Cardiovascular effect and stimulus-dependent inhibition of superoxide generation from human neutrophils by tibenelast, 5,6-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt (LY186655). 217 1

The venom from Crotalus molossus nigrescens contains many activities including: hyde powder azure proteinase; N-benzoyl-arginine-ethyl-ester hydrolase; phospholipase; phosphodiesterase; desoxyribonuclease; fibrinogen coagulase; collagenase, fibrinolytic activity, and hemorrhagic factors. The venom, assayed with amounts of venom up to 50 micrograms protein per assay, does not contain acetylcholinesterase, phosphatase, amylase, ribonuclease, tyrosyl-ester hydrolase or hyaluronidase activities. The venom is lethal to mice with an i.p. LD50 of 2.35 mg/kg mouse. Fractionation of soluble venom by Sephadex G-75 separates at least five families of components. Fractions I-III contains all the enzymes, and fraction V have six small peptides. Further separation of fractions II-III on diethyl-amino-ethyl-cellulose columns at pH 8.0 and 8.3 gave pure proteinase E with a mol. wt of 21,390 and the following N-terminal amino acid sequence; Phe-Ala-Lys-Arg-Tyr-Val-Glx-Leu-Val-Ile-Val-Ala. A thrombin-like enzyme with a mol. wt of 75,000 was also purified from this venom by means of affinity and ion exchange chromatographies.
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PMID:Characterization of the venom from Crotalus molossus nigrescens Gloyd (black tail rattlesnake): isolation of two proteases. 218 98


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