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)

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.
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PMID:Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli. 253 61

The stimulation of cultured guinea pig alveolar macrophages by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or by the phospholipid inflammatory mediator platelet activating factor (PAF) induced an increase in arachidonic acid release and its cyclooxygenase products. This release, which was mimicked by the association of threshold concentrations of the calcium ionophore A 23187 and of the protein kinase C activator tetradecanoyl phorbol acetate arose mainly from diacyl- and alkyl-acyl-phosphatidylcholine and phosphatidylinositol. Using [1-14C]arachidonic acid-labeled membranes as an endogenous substrate as well as dioleoyl-phosphatidyl [14C]ethanolamine as an exogenous substrate, we showed that phospholipase A2 activity of stimulated macrophages increases upon stimulation. Treatment of macrophages by prostaglandin E2 decreased the arachidonic acid release elicited by the chemotactic peptide and PAF. Furthermore, prostaglandin E2 increased and PAF decreased the cellular content in cyclic AMP. From these results we suggest that an initial stimulation of alveolar macrophages by a bacterial signal initiates the sequential activation of a phospholipase C and of phospholipase A2, leading to the release of PAF and eicosanoids. These mediators may in turn modulate the cell response by increasing or decreasing cyclic AMP, Ca2+, or diacyglycerol macrophage content.
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PMID:Phospholipase A2-mediated release of arachidonic acid in stimulated guinea pig alveolar macrophages: interaction with lipid mediators and cyclic AMP. 254 80

In order to analyze the complex activities of histamine H2 receptor activation on neutrophils, human HL-60 promyelocytic leukemia cells were differentiated into neutrophils by incubation with dimethyl sufoxide, loaded with the Ca2+-sensitive indicator dyes, indo-1 or fura-2, and the levels of intracellular Ca2+ ([Ca2+]i) measured in a fluorescent-activated cell sorter and fluorimeter, respectively. Histamine increased [Ca2+]i in a dose-dependent manner with a half-maximal concentration (EC50) of approximately 10(-6) to 10(-5) M, which exhibited H2 receptor specificity. Prostaglandin E2 and isoproterenol also induced [Ca2+]i mobilization in HL-60 cells, whereas the cell permeable form of cAMP and forskolin failed to increase [Ca2+]i. Since H2-receptor mediated [Ca2+]i mobilization was not inhibited by reducing the concentration of extracellular Ca2+ nor by the addition of Ca2+ channel antagonists, LaCl3 and nifedipine, [Ca2+]i mobilization is due to the release of Ca2+ from intracellular stores. Furthermore, both 10(-4) M histamine and 10(-6) M fMet-Leu-Phe increased the levels of 1,4,5-inositol trisphosphate. However, histamine-induced mobilization of [Ca2+]i was inhibited by cholera toxin but not by pertussis toxin, whereas the action of fMet-Leu-Phe was inhibited by pertussis toxin but not by cholera toxin. These data suggest that H2 receptors on HL-60 cells are coupled to two different cholera toxin-sensitive G-proteins and activate adenylate cyclase and phospholipase C simultaneously.
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PMID:Multiple signaling pathways of histamine H2 receptors. Identification of an H2 receptor-dependent Ca2+ mobilization pathway in human HL-60 promyelocytic leukemia cells. 255 5

Absolute concentrations of inositol phosphate isomers (InsP(s] were quantified in the myeloid cell line HL-60 using the metal-dye detection technique. Stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) led to distinct alterations in at least seven different inositol phosphate species. Whereas the intracellular concentrations of the tetrakisphosphate isomers (InsP4(s] were found below the micromolar range, inositol 1,3,4,5,6-pentakis- and hexakisphosphate levels were about two orders of magnitude higher (36 and 54 +/- 2 microM (mean +/- S.D.), respectively). The three InsP4(s) showed distinct kinetic pattern upon receptor activation, the transient elevation of inositol 1,3,4,5-tetrakisphosphate being faster both in onset and in redecrease than inositol 1,3,4,6-tetrakisphosphate. Whereas the two latter isomers reached maximally 2.75 and 2.9 +/- 0.2 microM, respectively, 1 min after stimulation, inositol 3,4,5,6-tetrakisphosphate remained elevated (3.5 +/- 0.4 microM) up to 5 min after fMLP. Unexpected changes in highly phosphorylated InsP(s) were observed, notably a rise in inositol 1,3,4,5,6-pentakisphosphate and in inositol hexakisphosphate to 52 +/- 3 and 60 +/- 1 microM, respectively. In terms of mass, the increases in highly phosphorylated inositols are by far highest among all InsP(s). Combining radiotracer method with mass determination it was observed that the specific radioactivity of various InsP(s) was different and changed markedly upon fMLP stimulation, in spite of a prolonged labeling period leading to apparent isotopic steady state. The data presented demonstrate agonist-induced elevations of highly phosphorylated InsP(s) and suggest that inositol 1,4,5-trisphosphate, product of receptor-activated phospholipase C, is metabolized rather via phosphorylation than only by dephosphorylation pathways.
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PMID:Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells. 255 10

Leukotriene E4 (LTE4) is shown to be a partial agonist of leukotriene D4 (LTD4) in differentiated U-937 cells. The data that support this conclusion are: 1) LTE4 completely displaced [3H]LTD4 from its receptors in U-937 cell membranes. 2) LTE4 induced only 30 +/- 4% of the maximal Ca2+ transient induced by LTD4 in the presence of 1 mM extracellular Ca2+ and 60 +/- 4% of the maximal LTD4 response in the absence of extracellular Ca2+. 3) LTE4 induced only a fraction of the inositol phosphates metabolized by LTD4. Moreover, LTE4 resulted in essentially no production of the inositol 1,4,5-trisphosphate isomer, while LTD4 induced a rapid and substantial transient increase in this isomer. The generation of inositol phosphates by both agonists was unaffected by extracellular Ca2+. 4) The EC50 values for Ca2+ mobilization for LTD4 and LTE4 corresponded with their affinity (Kd values) for the LTD4 receptor. 5) A series of structurally diverse LTD4 receptor antagonists blocked the Ca2+ mobilization responses to LTD4 and LTE4 with identical rank orders of potency. 6) LTE4 acted as an antagonist of LTD4 of potency. 6) LTE4 acted as an antagonist of LTD4 effects when they were coadministered. 7) LTE4 and LTD4 acutely desensitized Ca2+ mobilization to each other. All of the effects of LTE4 are explained by its partial agonist activity at the LTD4 receptor as shown by the following data. 1) Neither LTD4 nor LTE4 had any effect on the agonist activity of fMet-Leu-Phe, LTB4, or platelet-activating factor. 2) None of the above agonists or antagonists to the above receptors affected any of the activities of LTD4 or LTE4. 3) Neither LTD4 nor LTE4 induced desensitization of Ca2+ mobilization to any of the non-LTD4 receptor agonists tested. 4) Under the conditions studied, we have not observed any evidence of multiple subclasses of LTD4 receptors in U-937 cells. LTE4 is a partial agonist of the LTD4 receptor, because it can only couple the LTD4 receptor to a portion of the signaling system available to the receptor when occupied by LTD4. Specifically, LTD4 caused the activation of receptor-operated calcium channels, mobilization of intracellular Ca2+, the activation of phosphatidylinositol-phospholipase C, and the liberation of an additional, as yet undefined, intracellular mediator. To do this, LTD4 receptors couple to at least two and perhaps more guanine nucleotide binding proteins. LTE4 is unable to activate the phosphatidylinositol-phospholipase C but can mimic the other effects of LTD4.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanisms of leukotriene E4 partial agonist activity at leukotriene D4 receptors in differentiated U-937 cells. 255 34

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.
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PMID:Neomycin induces high-affinity agonist binding of G-protein-coupled receptors. 255 74

Murine bone marrow-derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor-1 (CSF-1) and granulocyte-macrophage CSF (GM-CSF). These macrophages can also be "activated," but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl-methionyl-leucyl-phenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c-myc mRNA and c-fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.
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PMID:Activation and proliferation signals in murine macrophages: relationships among c-fos and c-myc expression, phosphoinositide hydrolysis, superoxide formation, and DNA synthesis. 255 11

Receptor-bypassing stimulants of human polymorphonuclear leukocytes (PMNLs), such as ionomycin or phorbol 12-myristate 13-acetate (PMA), generate an increase in diacylglycerol (DAG) which is independent of a phospholipase C specific for phosphatidylinositol 4,5,-bisphosphate (PIP2). Activation of a phospholipase C specific for phosphatidylcholine (PC) has been implicated as a source of DAG in other cells by measuring the release of radiolabelled phosphorylcholine. However, since PMNLs could not be labelled sufficiently with [3H]choline, we developed an h.p.l.c. assay to quantify mass levels of phosphorylcholine after enzymic conversion to [32P]CDP-choline with CTP-phosphorylcholine (choline phosphate) cytidylyltransferase (EC 2.7.7.15). This assay was linear to at least 20 nmol, and was sensitive to 10 pmol of phosphorylcholine. Baseline phosphorylcholine levels in unstimulated PMNLs were 2300 +/- 510 pmol/10(7) cells and were decreased by pretreatment with PMA (166 nM) or ionomycin (1 microM) for 10 min by 360 +/- 130 and 600 +/- 290 pmol/10(7) cells respectively (P less than 0.05). In contrast, baseline DAG levels were 147.6 +/- 11.7 pmol/10(7) cells in unstimulated PMNLs, and were increased by PMA or ionomycin by 1320 +/- 222 and 1891 +/- 264 pmol/10(7) cells respectively (P less than 0.05). Similarly, the chemoattractant fMet-Leu-Phe raised DAG levels by 731 +/- 111 pmol/10(7) cells and decreased phosphorylcholine levels by 180 +/- 60 pmol/10(7) cells. Activation of PMNLs by PMA, ionophore or fMet-Leu-Phe thus leads to the sustained production of DAG accompanied by the disappearance of phosphorylcholine. This suggests that these stimulants enhance PC turnover via a hydrolytic mechanism which is independent of phospholipase C, with activation of a PC-specific phospholipase D being a plausible mechanism.
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PMID:Stimulation of phosphorylcholine turnover and diacylglycerol production in human polymorphonuclear leukocytes. Novel assay for phosphorylcholine. 276 12

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.
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PMID:Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape. 277 66

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubation with alkyl-[32P]lysoPC. Upon stimulation with the chemotactic peptide, formylMet-Leu-Phe (fMLP), these 32P-labeled cells produce 1-O-alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and, in the presence of ethanol, 1-O-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because the cellular ATP contains no 32P, alkyl-[32P]PA and alkyl-[32P]PEt must be formed from alkyl-[32P]PC by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Analyses of the sn-1 bonds by selective hydrolysis and mass measurements reveal that the PA and PEt formed during stimulation contain both ester and ether bonds with distributions similar to that in the endogenous PC. Furthermore, in neutrophils labeled in alkyl-[32P]PC, the specific activities of the diradyl-PA and diradyl-PEt formed during stimulation are similar to that of diradyl-PC. These results demonstrate that the fMLP-induced PLD utilizes diradyl-PC as the major substrate. It is further concluded that, at early times (30 s), PA and PEt are both formed almost exclusively by PLD. Following stimulation with fMLP, neutrophils double-labeled in alkyl-PC by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC generate [3H]alkyl-DG and [32P]orthophosphate [( 32P]PO4) with superimposable kinetics, indicating degradation of PA by a phosphohydrolase. Generation of [3H]alkyl-DG and [32P]PO4 lags behind PA formation and parallels the decline in PA accumulation. In addition, generation of both [3H]alkyl-PA and [3H]alkyl-DG requires extracellular Ca2+ and cytochalasin B. Furthermore, the phosphohydrolase inhibitor, propranolol, decreases both [3H]alkyl-DG and [32P]PO4 while increasing [3H]alkyl-PA and not altering [3H]alkyl-PEt. Moreover, the decreases in DG are accounted for by increases in PA. These results demonstrate that PLD-derived alkyl-PA is degraded by a phosphohydrolase to produce alkyl-DG. DG formed during stimulation contains both ester and ether-linked species and this DG formation is inhibited completely by propranolol. Upon stimulation, alkyl-[32P]PC-labeled neutrophils do not produce [32P]phosphocholine, suggesting that PC is not hydrolyzed by phospholipase C. In addition, PA is formed in amounts sufficient to account for all of the DG formed during stimulation. It is concluded that the DG formed during fMLP stimulation is derived almost exclusively from PC via the PLD/PA phosphohydrolase pathway.
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PMID:Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction. 279 44


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