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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells, which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered, the rank order of potency is oleoyl-LPA > arachidonoyl-LPA > linolenoyl-LPA > linoleoyl-LPA > stearoyl-LPA = palmitoyl-LPA > myristoyl-LPA. The shorter-chain species, lauroyl- and decanoyl-LPA, show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses > 15 microM. Similarly, the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate, a putative messenger with some structural similarities to LPA, elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however, cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that, although many features of the LPA structure are important for optimal activity, the phosphate group is most critical, suggesting that this moiety is directly involved in receptor activation.
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PMID:Lysophosphatidic acid-induced Ca2+ mobilization in human A431 cells: structure-activity analysis. 773 3

Recent studies indicate that the vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 exerts rapid effects (seconds to minutes) in a variety of cell types. These rapid nongenomic actions in osteoblasts include effects on membrane voltage-gated calcium channels, phospholipase C activity, and the sodium/hydrogen antiport. Since the rapid effects occur in osteoblasts that lack the nuclear vitamin D receptor, it is postulated that the nongenomic responses to the hormone reflect interaction with a separate, membrane localized signalling system. Preliminary studies demonstrate the presence of a receptor on the membranes of osteoblasts that lack the nuclear vitamin D receptor. This membrane receptor recognizes 1 alpha,25-dihydroxyvitamin D3 and its inaction 1 beta epimer, but not 25-hydroxyvitamin D3. These rapid nongenomic actions generated by interaction with the membrane receptor modulate the effects of the hormone on gene transcription. Thus, the rapid nongenomic pathway may play a regulatory function in modulating the genomic pathways affected by 1 alpha,25-dihydroxyvitamin D3.
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PMID:Nongenomic actions of the steroid hormone 1 alpha,25-dihydroxyvitamin D3. 787 22

A water-soluble, chemiluminescent substrate for sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) activity was synthesized. This was achieved by replacing the diacylglycerol moiety found in the natural substrate, phosphatidylinositol, by a dioxetane-containing moiety, resulting in the synthetic substrate (+/-)-3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo[3.3.1.1(3,7]decan]-4-yl)phenyl myo-inositol-1-O-hydrogen phosphate (LUMI-PI). PI-PLC-catalyzed cleavage of the phosphodiester bond of LUMI-PI results in the formation of a chemiluminescent precursor, the labile anion AMP-D, which decays with emission of light. An assay procedure was developed using 96-well microtiter plates with detection of the chemiluminescence on autoradiography film. With this procedure the detection limit of purified phospholipase C was about 10 pg. Furthermore, PI-PLC activity was readily detected in situ using small quantities (< 1 microliters) of liquid cultures of PI-PLC-producing bacterial strains.
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PMID:A chemiluminescent substrate for the detection of phosphatidylinositol-specific phospholipase C. 810 48

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.
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PMID:Role of hydroxyl radicals in the activation of human platelets. 817 49

The aim of this study was to investigate the stimulating effects of sulfhydryl reagents on glucose transport in isolated rat heart muscle cells and to compare them with the action of insulin. Low concentrations of the sulfhydryl oxidants hydrogen peroxide (H2O2) and diamide (5-100 microM), but also of phenylarsine oxide (PAO) (0.5-3 microM), that is known to specifically react with vicinal SH-groups, stimulated the rate of 2-deoxy-D-glucose uptake by a factor of 4 to 8 in these cells, while higher concentrations were inhibitory. The stimulating effects of H2O2 or diamide, and, to a significantly lesser extent, those of PAO or insulin, were depressed in cells pretreated with the sulfhydryl-alkylating agent N-ethylmaleimide (56-100 microM). H2O2 raised the Vmax and lowered the Km of 3-O-methyl-D-glucose uptake, while PAO or insulin solely increased Vmax. The increase in glucose transport caused by H2O2 was antagonized by the beta-adrenergic agonist isoprenaline (1 microM) or by a membrane-permeant cyclic AMP analog, whereas the effects of PAO or insulin were not altered. The action of H2O2 was additive with the stimulation induced by the protein phosphatase inhibitors okadaic acid (1 microM) or vanadate (6 mM), whereas the responses to PAO or insulin were reduced in the presence of these agents. Finally, H2O2 and PAO, but not insulin, acted additively with the protein kinase C ligand phorbol myristate acetate (0.8 microM) and with phospholipase C (0.03 units/ml). We conclude that, in cardiac myocytes, H2O2, on the one hand, and PAO (and possibly insulin), on the other hand, stimulate glucose transport via at least two distinct, SH-dependent pathways. These pathways, in turn, differ from a protein kinase C- and from a phospholipase C-mediated mechanism.
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PMID:Phenylarsine oxide and hydrogen peroxide stimulate glucose transport via different pathways in isolated cardiac myocytes. 824 Dec 56

We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels. The enzyme activity in rat cardiac sarcolemmal membranes that had been preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts superoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Such decrease was fully prevented by catalase. Preincubation of sarcolemma with hypochlorous acid also gave a significant inhibition of phospholipase C, which was counteracted by the synthetic thiol reducer dithiothreitol. H2O2-pretreatment induced a concentration-dependent inhibition of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H2O2 effects. These data suggest that superoxide anions and hydroxyl radicals did not interfere with phospholipase C activity, and that the nonradical oxidants, H2O2 and hypochlorous acid, may have acted through oxidation of thiol (SH) groups. The existence of reactive SH groups associated with the enzyme was confirmed by the inhibitory effects of SH modifiers (p-chloromercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide and methyl methanethiosulfonate), which were prevented and in some cases also reversed by dithiothreitol. The biological reducer glutathione (GSH) was not able to recover the H2O2-induced inhibition of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enzyme was active in a wide range of GSH/GSSG redox states, but H2O2 pretreatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deactivated the enzyme. The oxidants' concentrations that significantly impaired phospholipase C in this study were compatible with those occurring in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235, 1991]. This supports the possibility that alteration of the receptor-associated phospholipase C may be a factor in the oxidant-related dysfunction of the ischemic-reperfused heart.
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PMID:Oxidative stress modifies the activity of cardiac sarcolemmal phospholipase C. 828 Jul 55

We have reported that U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole- 2,5-dione) an inhibitor of phospholipase C-dependent processes in human polymorphonuclear neutrophils (PMN) and platelets, potently suppresses the responsiveness of suspended PMN and platelets to receptor agonists. We demonstrate here that U-73122 caused a concentration-dependent (10-800 nM) inhibition of N-formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha (TNF alpha), interleukin-8 and phorbol myristate acetate (PMA)-triggered PMN adhesion on fibronectin, fetal bovine serum or keyhole limpet hemocyanincoated microtiter plates. U-73122 also inhibited PMN adherence to and transmigration through TNF-alpha-activated endothelium (IC50 < 50 nM). Further, U-73122 suppressed interleukin-8, N-formylmethionyl-leucyl-phenylalanine and PMA-stimulated up-regulation of the beta 2-integrin, Mac-1 (CD11b/CD18), on the PMN surface (IC50 < 1.3 microM). U-73122 also caused a time-(15-120 min) and concentration-dependent inhibition (IC50 = 25-100 nM) of the N-formyl-methionyl-leucyl-phenylalanine-, TNF alpha- and PMA-elicited adhesion-dependent, oxidative burst, measured as hydrogen peroxide (H2O2) production, in PMN. The CD18-dependent extracellular release of lactoferrin from PMN activated with these stimuli was also suppressed by U-73122. U-73343 (1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine dione), a close analog of U-73122, did not affect PMN responsiveness.
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PMID:U-73122: a potent inhibitor of human polymorphonuclear neutrophil adhesion on biological surfaces and adhesion-related effector functions. 876 66

We measured the ability of sphingomyelin (SPM) to inhibit phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis catalyzed by human phospholipase C-delta 1 (PLC-delta 1) in model membranes and detergent phospholipid mixed micelles. SPM strongly inhibited PLC-delta 1 catalytic activity measured in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (PC), PI(4,5)P2, and SPM from brain or egg. At 37 or 45 degrees C, the rate of PI(4,5)P2 hydrolysis in PC/SPM/PI(4,5)P2 vesicles (15:80:5 mol:mol) was less than 25% of that observed in PC/PI(4,5)P2 vesicles (95:5). By contrast, catalysis was only weakly inhibited by equivalent concentrations of the SPM analog, 3-deoxy-2-O-stearoyl-SPM, which lacks hydrogen bond-donating groups at the C-3 and C-2 positions of the sphingolipid backbone. Inhibition by SPM was not observed in detergent/phospholipid mixed micelles. The binding affinity of PLC-delta 1 for vesicles containing PC and PI(4,5)P2 was slightly diminished by inclusion of SPM in the lipid mixture, but not enough to account for the decreased rate of catalysis. We could find no evidence of specific binding of the enzyme to SPM, which argues against a simple negative allosteric mechanism. To understand the cause of inhibition, the effects of SPM and 3-deoxy-2-O-stearoyl-SPM on the bulk properties of the substrate bilayers were examined. Increasing the mole fraction of SPM altered the fluorescence emission spectra of two sets of head group probes, 6-lauronyl(N,N-dimethylamino)naphthalene and N-[5-(dimethylamino)naphthalene-1-sulfonyl]-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, that are sensitive to water content at the membrane/solution interface. Results obtained with both probes suggested a reduction in hydration with increasing SPM content. Vesicles containing 3-deoxy-2-O-stearoyl-SPM produced intermediate changes. Our results are most consistent with a model in which SPM inhibits PLC by increasing interlipid hydrogen bonding and by decreasing membrane hydration; both factors raise the energy barrier for activation of PLC-delta 1 at the membrane/protein microinterface.
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PMID:Inhibition of phospholipase C-delta 1 catalytic activity by sphingomyelin. 894 52

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.
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PMID:Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling. 897 7

In the yeast two-hybrid system, a 100-residue fragment (beta1A) from the N terminus of the beta1 subunit interacts with domains specific to adenylyl cyclase 2 (AC2), the muscarinic atrial potassium channel (GIRK1), and phospholipase C-beta2 (PLC-beta2). Based on the crystal structure of the G protein, beta1A is composed of an N-terminal alpha helix, a loop, and five beta strands in which the C-terminal four beta strands form a beta sheet, the first of seven sheets that make up the propeller structure of the beta subunit. A mutant of beta1A (L4P, L7P, and L14P), in which the alpha helix was potentially destroyed, interacted poorly with the G protein gamma subunit but effectively with domains of AC2, GIRK1, and PLC-beta2. In contrast, another mutant of beta1A (S72A, D76A, and W82A), in which a network of hydrogen bonds was disrupted, interacted poorly with GIRK1 and PLC-beta2 domains, but effectively with the gamma subunit and the AC2 domain. These results suggest that the proper folding of the first five beta strands in the G protein beta subunit is a requirement for appropriately positioning residues that interact with GIRK1 and PLC-beta2. Furthermore, since mutations that potentially disrupted the folding of these beta strands did not affect interaction with AC2, the structural determinants on the G protein beta subunit for interaction with various effectors may be different.
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PMID:Structural determinants for interaction with three different effectors on the G protein beta subunit. 899 2


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