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)

Proximal tubules were isolated in highly pure form from rabbit cortices by a mechanical procedure that is known to preserve the structural and metabolic aspects of the tubular cells. Postnuclear supernates prepared from the isolated tubules were subjects to isopycnic centrifugation in linear sucrose gradients. The enzyme activities associated with the plasma membrane (gamma-glutamyl transpeptidase, amino-peptidase M, alkaline phosphatase, Na-K-ATPase, and phosphodiesterase I) exhibited sharp unimodal frequency-density profiles with a median density near 1.16 g/ml, which shifted to a heavier density when treated with digitonin. The lysosomal enzymes, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and cathepsin B, and the peroxisomal enzyme catalase exhibited particle-associated activity near a density of 1.22 g/ml. Disruption of these particles by freezing and thawing resulted in these activities appearing in the rho = 1.10 g/ml region of the gradient where the soluble cytosolic enzyme, phosphoglucomutase, exhibited activity. Cytochrome oxidase activity typical of mitochondria gave a sharp unimodal profile at rho = 1.18 g/ml. Microsomal glucose-6-phosphatase and NADPH: cytochrome c reductase activities gave median densities near 1.16 g/ml, which did not change after incubation with digitonin. Galactosyl transferase activity gave a skewed profile at rho = 1.16 g/ml and showed a slight shift to heavier density after digitonin. This study of the enzymatic activities and density gradient distribution of the components of the proximal tubule cells provides the methodology for the further study of the cellular processing of endogenous and exogenous substances by this vital cell type.
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PMID:Analytical cell fractionation of isolated rabbit renal proximal tubules. 730 Jan 16

Sensitization by 1-methyl-3-isobutylxanthine (MIX), a potent inhibitor of cAMP phosphodiesterase, of cellular sensitivity to mitomycin C (MC) was tested using various cell lines. Sensitization was seen with CHO cells and Balb/c 3T3 cells, but a decrease in sensitivity was seen with HeLa, K-balb and other cell lines. To measure the extent of crosslinks produced by MC, we used an alkaline elution assay. The extent of crosslinks was increased by MIX in CHO cells and decreased in K-balb cells. These changes in extent are well reflected by changes in sensitivity to MC in both cell lines. In CHO cells, an intracellular dose of MC was increased by MIX with no change in the rate of uptake or efflux of MC. There was a slight decrease in the dose in HeLa or K-balb cells. Among the enzymes which engage in the reductive activation of MC, we tested DT-diaphorase and NADPH-cytochrome P450 reductase using CHO, HeLa and K-balb cells. MIX had almost no effect on the activity of NADPH-cytochrome P450 in each cell line, whereas it suppressed the activity of DT-diaphorase, significantly in HeLa and K-balb cells, and slightly in CHO cells. All these facts indicate that MIX caused an increase in the extent of crosslinks via an increase in the intracellular dose of MC in CHO cells, and that these increases may lead to the sensitization. On the other hand, the decrease in the sensitivity shown in HeLa and K-balb cells may be due to the suppression of DT-diaphorase activity and/or to a decrease in MC dose.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cell line-dependent changes in sensitivity to mitomycin C by 1-methyl-3-isobutylxanthine is due to an altered intracellular dose of the anticancer drug and/or to changes in DT-diaphorase activity. 753 Oct 52

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.
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PMID:ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803. 776 63

Nimesulide, the prototype of a new class of anti-inflammatory drugs, dose-dependently decreases the production of the superoxide anion (O2-.) in N-formyl-methionyl-leucyl-phenylalanine (fMLP)- and in phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes. The inhibition of O2-. is possibly related to its inhibitory effect on polymorphonuclear leukocyte cytosolic phosphodiesterase type IV (IC50 = 39 +/- 2 microM), to the related increase in cAMP (P < 0.01 at 1 microM) and the subsequent increase in protein kinase A activity. In fact H-89, a specific protein kinase A inhibitor, counteracts the inhibitory effect of nimesulide on O2-. production by fMLP and PMA. The activation of protein kinase A may prompt the phosphorylation of a number of substrates, thus inhibiting the assembly of NADPH-oxidase in the plasma membrane. Accordingly, nimesulide decreases PMA-induced assembly of NADPH-oxidase in polymorphonuclear leukocytes plasma membranes by about 35%. Protein kinase A activation may also interfere with chemotaxis. Nimesulide inhibits stimulated chemotaxis and the effect is decreased by H-89. Inhibition of phosphodiesterase type IV may explain many of nimesulide's effects.
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PMID:Nimesulide decreases superoxide production by inhibiting phosphodiesterase type IV. 780 66

An enzymatic assay for adenosine 3':5'-monophosphate (cAMP) is described. Current measurement techniques can be expensive, time-consuming, and lack versatility. The critical step of this new method is the enzymatic destruction of endogenous purinergic noncyclic nucleotides. The diester linkage of cAMP is then cleaved and AMP is phosphorylated to ATP. Newly formed ATP is amplified using ATP-ADP cycling reactions and NADPH is measured fluorometrically. cAMP was measured in neonatal rat ventricular myocytes cultured on standard 100-mm dishes and treated with 2 microM 3-isobutyl-1-methylxanthine +/- 1 microM isoproterenol. When the enzymatic fluorometric assay was compared with an immunocolorimetric assay and a radioimmunoassay, cAMP content (pmol/plate mean +/- SE) was 124.3 +/- 6.7, 130.6 +/- 3.9, and 144.0 +/- 4.4 without isoproterenol and 656.4 +/- 23.5, 659.5 +/- 54.1, and 677.1 +/- 48.9 with isoproterenol, respectively. The standard curve with the enzymatic fluorometric assay is linear, in contrast to the curves of the nonlinear immunocolorimetric assay and radioimmunoassay. The enzymatic fluorometric assay can be used to detect < 20 fmol of cAMP/sample and can be adapted to measure < 1 fmol/sample. It can also be used to measure the activities of adenylate cyclase and phosphodiesterase. In summary, this enzymatic cAMP assay is sensitive, safe, versatile, and inexpensive and has multiple potential applications.
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PMID:An enzymatic fluorometric assay for adenosine 3':5'-monophosphate. 805 55

Reactive oxygen-derived free radical species have been implicated in the pathogenesis and pathophysiology of inflammatory lung diseases. In a guinea pig model of aerosolized endotoxin-induced bronchial hyperresponsiveness to substance P, a possible involvement of oxidative lung injury was assessed by measuring the changes in membrane-bound neutral endopeptidase activity in the airway tissues and the level of lipid peroxides in the plasma. Vehicle-treated animals developed a neutrophilic airway inflammation, bronchial hyperresponsiveness to substance P associated with neutral endopeptidase hypoactivity, and elevation of lipid peroxides at 18 to 24 h after an exposure to endotoxin (75 microgram/ml, 40 min). A nonselective phosphodiesterase inhibitor, aminophylline, and selective phosphodiesterase isoenzyme inhibitors, SDZ-ISQ-844 (type III/IV) and SDZ-MKS-492 (type III), attenuated the neutrophilic airway inflammation induced by endotoxin. Aminophylline, SDZ-MKS-492, and a superoxide anion-generating NADPH-oxidase inhibitor apocynin inhibited bronchial hyperresponsiveness to substance P with attenuation of neutral endopeptidase inactivation induced by endotoxin. SDZ-ISQ-844, SDZ-MKS-492, and apocynin attenuated the elevation of lipid peroxides. The generation of hypochlorite (OCl-) from whole blood leukocytes was attenuated by aminophylline, SDZ-ISQ-844, SDZ-MKS-492, and apocynin at 1 to 2 h after exposure. These results suggest that reactive oxygen-derived free radical species-mediated oxidative lung injury may play an important role in endotoxin-induced bronchial hyperresponsiveness to substance P, and that phosphodiesterase isoenzyme inhibitors may be potentially useful as anti-inflammatory drugs.
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PMID:A possible involvement of oxidative lung injury in endotoxin-induced bronchial hyperresponsiveness to substance P in guinea pigs. 970 1

The effects were studied of the non-specific phosphodiesterase inhibitor, theophylline (37.5-300 microM), on intracellular levels of cyclic adenosine monophosphate (cAMP) and superoxide generation following exposure of human neutrophils to four different stimuli of neutrophil membrane-associated oxidative metabolism, each of which utilizes a different transductional mechanism to activate NADPH-oxidase, in vitro. Exposure of neutrophils to FMLP (1 microM), the calcium ionophore A23187 (1 microM), and opsonized zymosan (OZ, 0.5 mg/ml) was accompanied by activation of superoxide production and increased concentrations of intracellular cAMP. Inclusion of theophylline resulted in augmentation of cAMP and inhibition of superoxide production by these stimuli. These negative effects of theophylline on neutrophil superoxide generation were mimicked by dibutyryl cAMP and 8-bromo-cGMP, while the inhibitory activity of all 3 agents was antagonized by the protein kinase A inhibitor KT 5720, but not by the G-kinase inhibitor KT 5823. Unlike FMLP, OZ and A23187, intracellular cAMP levels did not increase in cells activated with phorbol-12-myristate-13-acetate (PMA, 25 ng/ml), while oxidant production activated by this stimulus was insensitive to the inhibitory effects of theophylline. These observations suggest that the beneficial, anti-inflammatory interactions of theophylline with human neutrophils are related to the phosphodiesterase inhibitory properties of this agent, and are selective for those pro-inflammatory stimuli which elevate cAMP, resulting in enhanced activity of protein kinase A and inhibition of the production of potentially harmful reactive oxidants by these cells.
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PMID:Anti-oxidative effects of theophylline on human neutrophils involve cyclic nucleotides and protein kinase A. 982 70

The calcium/calmodulin-dependent activation of nitric-oxide synthase (NOS) and its production of nitric oxide (NO) play a key regulatory role in plant and animal cell function. SCaM-1 is a plant calmodulin (CaM) isoform that is 91% identical to mammalian CaM (wild type CaM (wtCaM)) and a selective competitive antagonist of NOS (Cho, M. J., Vaghy, P. L., Kondo, R., Lee, S. H., Davis, J. P., Rehl, R., Heo, W. D., and Johnson, J. D. (1998) Biochemistry 37, 15593-15597). We have used site-directed mutagenesis to show that a point mutation, involving the substitution of valine for methionine at position 144, is responsible for SCaM-1's inhibition of mammalian NOS. An M144V mutation in wild type CaM produced a mutant (M144V) which exhibited nearly identical inhibition of NOS's NO production and NADPH oxidation, with a similar K(i) (approximately 15 nM) as SCaM-1. A V144M back mutation in SCaM-1 significantly restored its ability to activate NOS's catalytic functions. The length of the hydrophobic amino acid side chain at position 144 appears to be critical for NOS activation, since M144L and M144F activated NOS while M144V and M144C did not. Despite their competitive antagonism of NOS, M144V, like SCaM-1, exhibited a similar dose-dependent activation of phosphodiesterase and calcineurin as wtCaM. SCaM-1 and M144V produced greater inhibition of NOS's oxygenase domain function (NO production) than its reductase domain functions (NADPH oxidation and cytochrome c reduction). Thus, CaM's methionine 144 plays a critical role the activation of NOS, presumably by influencing the function of NOS's oxygenase domain.
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PMID:A point mutation in a plant calmodulin is responsible for its inhibition of nitric-oxide synthase. 1059 8

The relationship between activation of NADPH-oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store-operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 microM) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH-oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.
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PMID:Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease. 1120 56

1. The metabolism of a novel phosphodiesterase-IV inhibitor (V11294) was studied in human liver microsomal and cytosol preparations and in cDNA-expressed human hepatic CYP forms. 2. Human liver microsomes, but not cytosol, catalysed the NADPH-dependent metabolism of V11294 to V10331 (formed by hydroxylation of the cyclopentyl ring), V10332 (N-desethyl V11294) and V11689 (formed by hydroxylation of the isopropyl side chain). In addition, smaller amounts of a secondary metabolite V11690 (which can be formed from either V10332 or V11689) were also produced. 3. Kinetic analysis of V11294 metabolism to V10331, V10332 and V11689 in two preparations of pooled human liver microsomes revealed average K(m) = 2.5, 8.1 and 3.9 micro M, respectively. 4. The metabolism of V11294 was determined with a characterized bank of 16 individual human liver microsomal preparations employing a V11294 substrate concentration of 8 micro M (i.e. approximately the K(m) for V10332 formation and around twice the K(m) for V10331 and V11689 formation). Good correlations (r(2) = 0.570-0.903) were observed between V10331, V10332 and V11689 formation and markers of CYP3A forms. In contrast, poorer correlations (r(2) = 0.0002-0.428) were observed with markers of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP forms, V11294 (8 micro M) was metabolized by cDNA-expressed CYP3A4 to V10331, V10332 and V11689, with lower amounts of V11690 also being formed. Lower rates of V11294 metabolism to some V11294 metabolites were also observed with cDNA-expressed CYP2C9, CYP2C19 and CYP2D6, whereas only very low or undetectable rates of V11294 metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8 and CYP2E1. 6. The metabolism of V11294 (8 micro M) to V10331, V10332 and V11689 was markedly inhibited by the CYP3A mechanism-based inhibitor troleandomycin. In contrast, V11294 metabolism was not significantly affected by inhibitors of CYP1A2, CYP2C9, CYP2D6 and CYP2E1 or by the CYP2C19 substrate S-mephenytoin. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, V11294 metabolism in human liver to V10331, V10332 and V11689 appears to be primarily catalysed by CYP3A forms.
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PMID:Metabolism of a novel phosphodiesterase-IV inhibitor (V11294) by human hepatic cytochrome P450 forms. 1216 Apr 84


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