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)

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.
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PMID:Agonist and antagonist properties of calmodulin fragments. 632 72

A continuous spectrophotometric assay for cAMP phosphodiesterase has been optimized and adopted for assaying calmodulin in biological samples. This method utilizes the coupled enzyme reactions of myokinase, pyruvate kinase, and lactic acid dehydrogenase. The effective molar extinction coefficient for this method is 1.25 X 10(4) at 340 nm. A point-assay method capable of handling a large number of samples has also been established. This same procedure can also be adopted for the assay of calcineurin and other calmodulin-binding proteins.
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PMID:An optimized continuous assay for cAMP phosphodiesterase and calmodulin. 632 36

Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin, PP2B) of Saccharomyces cerevisiae is implicated in adaptation to high-salt conditions. Calcineurin mediates high salt-induced expression of the ENA1/PMR2 gene encoding the P-type ATPase, which is suggested to be involved in Na+ efflux. We identified the PDE1 gene encoding the low-affinity cAMP phosphodiesterase as a multicopy suppressor of the Li(+)- and Na(+)-sensitive calcineurin null mutant, suggesting that cAMP is a negative regulator of adaptation to high-salt stress. Genetic analysis indicated that calcineurin and cAMP act antagonistically in a common pathway for adaptation. The bcy1 disruption, which leads to constitutive cAMP-dependent protein kinase (PKA) activity inhibited high NaCl-induced expression of the ENA1/PMR2 gene, caused an elevation of the intracellular Na+ level and a growth defect in high-NaCl medium, all of which were analogous to the defects of a calcineurin mutant. A reduced cAMP level resulting from multiple copies of the PDE1 gene caused increased expression of the ENA1/PMR2 gene in response to high NaCl. We propose a model for the regulation of cation homeostasis, in which calcineurin antagonizes PKA to activate transcription of the ENA1/PMR2 gene in response to high-salt conditions.
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PMID:Adaptation to high-salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase. 750 Sep 49

The role of phosphoprotein phosphatase in the regulation of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rat pinealocytes was investigated using the three phosphatase inhibitors calyculin A, tautomycin, and okadaic acid. Calyculin A (0.1 microM) was found to enhance the isoproterenol- and norepinephrine-stimulated cAMP accumulation six- and threefold, respectively, whereas tautomycin and okadaic acid were less effective. The effect of calyculin A was rapid (within 5 min) and persisted in the presence of phosphodiesterase inhibition. However, in contrast to protein kinase C activation or intracellular calcium elevation, the phosphatase inhibitors were less effective in potentiating the cAMP response stimulated by forskolin or cholera toxin, and their effects were not blocked by calphostin C or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. The adrenergic-stimulated cGMP response was also less sensitive to the phosphatase inhibition. Therefore, our results suggest that 1) the adrenergic-stimulated cAMP signal is subjected to the tonic inhibition by phosphoprotein phosphatase; 2) phosphatase inhibitors enhance cAMP synthesis through their actions at the receptor level; and 3) the cAMP signal is more sensitive to the regulation by phosphorylation than cGMP in rat pinealocytes.
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PMID:Phosphatase inhibitors potentiate adrenergic-stimulated cAMP and cGMP production in rat pinealocytes. 753 89

Protein phosphatases regulate the activity of signal transduction mechanisms by dephosphorylating activated components. By utilizing selective inhibitors of these phosphatases, we investigated their role in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line. PTHrP, PTH and PGE2 stimulated cAMP accumulation up to 100-fold. Calyculin A, a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A (PP2A), did not affect basal levels of cAMP, but concentrations of 10(-11) M to 10(-8) M increased PTHrP-, PTH-, and PGE2-stimulated cAMP accumulation up to 1.7-fold, and this increase was concentration-dependent. Similar results were obtained with tautomycin, another potent inhibitor of PP1 and PP2A. In contrast, okadaic acid, a potent inhibitor of PP2A which inhibited PP1 less potently, did not enhance PTHrP-, PTH-, or PGE2-stimulated cAMP accumulation. The effect of calyculin A on agonist-stimulated cAMP accumulation persisted in cells treated with isobutyl methylxanthine, a phosphodiesterase inhibitor. When the effect of calyculin A was compared with that of 4 beta-phorbol 12-myristate 13-acetate (PMA), it was found that while PMA enhanced both the receptor and forskolin-stimulated cAMP accumulation, calyculin A had no effect on the forskolin-stimulated cAMP accumulation. The effect of calyculin A on PTHrP- and PTH-stimulated cAMP accumulation persisted in cells treated with PMA. These results suggest that protein phosphatases play an important role in agonist-stimulated cAMP accumulation in osteoblast-like cells, and that PP1 but not PP2A may be the major phosphatase involved. In contrast to activation by protein kinase C, the site of action for the phosphatase appears to be predominantly at a step prior to the activation of adenylyl cyclase in the cAMP signal transduction pathway.
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PMID:Inhibition of serine/threonine protein phosphatases enhances agonist-stimulated cAMP accumulation in UMR 106 osteoblast-like cells. 754 25

Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 mumol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 mumol/L). When ICa(L) was enhanced by Bay K8644 (1 mumol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n = 36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 mumol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.
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PMID:cGMP-dependent protein kinase regulation of the L-type Ca2+ current in rat ventricular myocytes. 755 27

Inorganic lead inhibits neurite initiation in cultured rat hippocampal neurons at concentrations as low as 100 nM. Conflicting reports suggest that Pb2+ may stimulate or inhibit protein kinase C, adenylyl cyclase, phosphodiesterase, and calmodulin, or increase intracellular free Ca2+ concentrations. Therefore, Pb2+ may alter the activities of Ca2+/calmodulin-dependent protein kinase (CaM kinase) or protein kinases C or A. We cultured rat hippocampal neurons in 100 nM PbCI2 alone or in combination with kinase or calmodulin inhibitors. Inhibiting protein kinase C with calphostin C exacerbated the inhibition of neurite initiation caused by PbCI2, but inhibiting protein kinase A with KT5720, CaM kinase with KN62, or calmodulin with calmidazolium completely reversed the effects of PbCI2. These results indicate that Pb2+ may inhibit neurite initiation by inappropriately stimulating protein phosphorylation by CaM kinase or cyclic AMP-dependent protein kinase (PKA), possibly by stimulating calmodulin. This hypothesis is supported by findings that other treatments that should increase protein phosphorylation (okadaic acid, a protein phosphatase inhibitor, and Sp-cAMPS, a PKA activator) also reduced neurite initiation. Whole-cell intracellular free Ca2+ ion concentrations were not significantly altered by 100 nM PbCI2 at 4, 12, 24, or 48 hr. Therefore, the hypothesized stimulatory effects of Pb2+ exposure on calmodulin, CaM kinase, or PKA are probably not caused by increases in whole-cell intracellular free Ca2+, but may be attributable either to intracellular Pb2+ or to localized increases in [Ca2+]in that are not reflected in whole-cell measurements.
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PMID:Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation. 767 45

The influence of internal Ca2+ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca2+ current (ICa). Internal free Ca2+ ([Ca2+]i) was varied between 0.01 and 10 microM by addition of Ca(2+)-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca2+]i depressed the 5-HT effect. The dose/effect curve for the Ca2+ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants Kd1 = 0.063 microM and Kd2 = 1 microM. Addition of calmodulin (CM) antagonists (50 microM trifluoperazine or 50 microM chlorpromazine), phosphodiesterase (PDE) antagonists [100 microM isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 microM okadaic acid (OA)] in the perfusion solution caused "anticalcium" action and modified the Ca2+ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca2+ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with Kd1 = 0.04 microM and Kd2 = 0.69 microM were obtained describing the activation of the retained unblocked enzyme--PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 microM), a blocker of Ca(2+)-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca(2+)-induced suppression of the cAMP-dependent upregulation of Ca2+ channels is due to involvement of two Ca(2+)-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca2+ concentration of less than or equal to 10 microM.
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PMID:Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones. 768 96

We have stably introduced expression vectors for the glucocorticoid receptor and a sensitive, hormone-responsive reporter (mouse mammary tumor virus-luciferase) into a human breast carcinoma-derived cell line. Employing this cell line, we have conducted a detailed examination of the induction of glucocorticoid-regulated genes and the phosphorylation of glucocorticoid receptor following pharmacologic manipulation of cell signaling pathways. The hormone response can be enhanced from 2 to 10-fold by activators of protein kinase A, protein kinase C, and inhibitors of protein phosphatase. Forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP), but not BrcGMP, enhance the hormone effect, yet surprisingly, phosphodiesterase inhibitors, isobutylmethylxanthine and Ro20-1724, strongly inhibit hormone-mediated induction of the reporter gene. These treatments do not alter cellular receptor content, dexamethasone binding, nor hormone-mediated receptor down-regulation. Tryptic peptide analysis of 32P-labeled receptor reveals that neither BrcAMP, isobutylmethylxanthine, nor the tumor promoter and protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate, detectably alter the state of glucocorticoid receptor phosphorylation. The only agent which alters receptor phosphorylation is the protein phosphatase inhibitor okadaic acid, but only at concentrations higher than required for maximum effects on glucocorticoid receptor transactivation. We propose that these effectors do not modify receptor directly but alter its interaction with transcription complexes.
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PMID:Modulation of cell signaling pathways can enhance or impair glucocorticoid-induced gene expression without altering the state of receptor phosphorylation. 769 81

In this study we have evaluated the mechanisms mediating the prolonged hyperalgesia induced by administration of prostaglandin E2 plus rolipram, an inhibitor of type IV phosphodiesterase. The Randall-Selitto paw pressure device was employed to measure the effect of intradermal injection of test agents on the time course of the decrease in mechanical nociceptive threshold produced by prostaglandin E2 plus rolipram in the hairy skin of the hindpaw of the rat. The intradermal injection of prostaglandin E2 produced a dose-dependent decrease in the nociceptive threshold which lasted approximately 2 h. While rolipram alone had no significant effect on nociceptive threshold, it enhanced and prolonged (> 72 h) prostaglandin E2-induced hyperalgesia. WIPTIDE, a protein kinase A inhibitor, when administered 30 min after prostaglandin E2, or with prostaglandin E2 plus rolipram, a time when prostaglandin E2-induced hyperalgesia was at its peak, produced a significant reduction in hyperalgesia. However, at 90 or at 180 min after injection of prostaglandin E2 plus rolipram, WIPTIDE was found to be without effect. H-8, a protein kinase G inhibitor, and okadaic acid, a protein phosphatase inhibitor, when administered 30 min after prostaglandin E2, or 180 min after prostaglandin E2 plus rolipram, produced no significant effect. However, when administered 90 min after prostaglandin E2 plus rolipram, each produced a significant reduction in the hyperalgesia induced by prostaglandin E2 plus rolipram.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Multiple second messenger systems act sequentially to mediate rolipram-induced prolongation of prostaglandin E2-induced mechanical hyperalgesia in the rat. 771 85


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