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)

1. The ability of acetylshikonin to inhibit the respiratory burst in rat neutrophils was characterized and the underlying mechanism of action was also assessed in the present study. 2. Acetylshikonin caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.48 +/- 0.03 and 0.39 +/- 0.03 microM, respectively. Acetylshikonin also inhibited the O2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3. Acetylshikonin did not scavenge the generated O2.- in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O2.- generation in these cell-free oxygen radical generating systems. 4. Acetylshikonin inhibited the formation of inositol trisphosphate (IP3) (39.0 +/- 7.8% inhibition at 10 microM, P < 0.05) in neutrophils in response to fMLP. 5. Both the neutrophil cytosolic protein kinase C (PKC) activity and the PMA-induced PKC associated with the membrane were unaffected by acetylshikonin. 6. Acetylshikonin did not affect the porcine heart protein kinase A (PKA) activity. Upon exposure to acetylshikonin, the cellular cyclic AMP level was decreased in neutrophils in response to fMLP. 7. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP/CB were inhibited by acetylshikonin (60.1 +/- 7.3 and 63.2 +/- 10.5% inhibition, respectively, at 10 microM, both P < 0.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 microM). 8. In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O2.- generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47phox in PMA-activated neutrophils (about 60% inhibition at 1 microM). 9. Collectively, these results suggest that the attenuation of protein tyrosine phosphorylation and a failure in the assembly of a functional NADPH oxidase complex probably contribute predominantly to the inhibition of respiratory burst in neutrophils by acetylshikonin. In contrast, the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways play only a minor role in this respect.
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PMID:Investigation of the inhibition by acetylshikonin of the respiratory burst in rat neutrophils. 917 81

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2.-/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 microM) or histamine (100 microM). Superoxide dismutase (50 U/ml), which dismutates O2.- into H2O2 also had no influence, whereas catalase (50 U/ml), which removes H2O2, completely diminished transient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 microM were used. Buffering trace amounts of iron (o-phenanthroline; 200 microM) in order to inhibit .OH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca(2+)-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca(2+)-ATPases of the endoplasmatic reticulum with thapsigargin (1 microM) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 microM) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O2.- or .OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological 'oxidative stress' associated with a progressive increase in [Ca2+]i.
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PMID:Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: role of hydrogen peroxide. 920 90

Silica is a well-known occupational fibrogenic agent and its primary target cell is alveolar macrophage. Particle-stimulated macrophages are believed to release various mediator which can regulate the inflammation as well as pulmonary fibrosis. Even though oxygen radicals play the major role among these mediators, the mechanisms concerning the stimulation of alveolar macrophages are not clear yet. The present study was carried out to investigate the signal transduction pathway on oxygen radical generation in silica-stimulated alveolar macrophages. Silica induced oxygen radical generation in a dose-response pattern. Extracellular calcium depletion, calcium channel blockers, and calcium release blocker decreased the effect of silica on oxygen radical generation. Silica increased intracellular calcium through the influx of calcium through the calcium channel and the calcium release from the intracellular calcium store. To know the role of protein kinase C (PKC), phospholipase C (PLC), and protein tyrosine kinase (PTK) in silica-induced oxygen radical generation, we pretreated alveolar macrophages with inhibitors of these enzymes. Inhibitors of PKC (sphingosine and staurosporine), PLC (neomycin and U-73122), and PTK (genistein and erbstatin) suppressed the silica-induced oxygen radical generation. Silica increased the PLC activity at the concentration of 5 mg/ml. The inhibitors of PTK and PLC suppressed the action of silica on the PLC activity. From these results, we suggest that silica induces oxygen radical generation through PTK, PLC, and PKC in alveolar macrophages.
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PMID:Silica-induced oxygen radical generation in alveolar macrophage. 924 22

The role of polymorphonuclear leukocytes (PMN) in stemming systemic infection is executed mainly by the utilization of molecular O2 leading to the production of reactive oxygen intermediates (ROI). PMN-derived ROI also serve as intra- and extracellular second messengers providing both positive and negative feedback on cellular autoregulation. We investigated the effect of endogenous ROI on two signal transducing pathways: the receptor (R)-G-protein-phospholipase D (PLD) and receptor (R)-G-protein-phospholipase C pathways responsible for the subsequent interleukin-8 (IL-8)-induced PMN respiratory burst. Purified human PMN were primed with LPS adhered to plastic surfaces and stimulated with IL-8 with or without the presence of each of five different selective ROI scavengers/antioxidants: DMSO, N(a)N3, L-alanine, catalase, or superoxide dismutase. Total IL-8 surface receptor expression was assessed by 125I-IL-8 and 125I-labeled mAbs against IL-8R type A and B binding assays; PLD activation was assessed by measuring formation of phosphatidyl ethanol (PEt) in the presence of ethanol; PLC activation was measured by quantitative conversion of [32P]ATP-labeled phosphatidic acid (PA) into diacylglycerol (DAG); expression of G alpha-inhibitory subunit was assessed by SDS-PAGE and immunoblotting with polyclonal Abs against this subunit. Production of O2-, H2O2, HClO, and myeloperoxidase (MPO) in the experimental model was confirmed in a separate set of experiments. The overall impact of antioxidants on each component of the transducing tripartite complex was stimulatory; however, N(a)N3 and SOD exhibited the most ubiquitous effect with consistent up-regulation by N(a)N3 of IL-8R expression, whereas even trace amounts of externally added authentic MPO significantly down-regulated the functional activity of both effector enzymes. These results demonstrate a multiple site-specific targeting of the signal-transducing complex by endogenous PMN-derived ROI and an overall protective effect of ROI scavengers/antioxidants.
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PMID:Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal transduction. 926 41

Several physiological agonists that induce elevation of cytosolic free calcium (Ca2+)-levels act via receptor coupled G-proteins, involving activation of phospholipase C (PLC) and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Activation of the inositol signal transduction pathway that precedes Ca2+ ion mobilization is a well accepted signaling pathway in endothelial cell eicosanoid synthesis. This study was designed to examine possible involvement of phosphoinositides in the effects of oxygen free radicals on Ca2+ liberation and eicosanoid synthesis in human umbilical venous endothelial cells (HUVEC). Hydrogen peroxide (H2O2) was chosen as oxygen radicals generating agent. Stimulation of HUVEC with H2O2 (0.1 mmol/l) led to significant rises in inositol phosphate and diacylglycerol (DAG) levels within 300 seconds and an inhibition of Ca2+ release from internal stores. Eicosanoid formation was detectable despite unchanged levels of cytosolic free Ca2+ and no detectable activation of membrane associated phospholipase A2 (PLA2). This suggests that eicosanoid formation may be mediated through the activation of a Ca2+ independent, cytosolic 40 kDa PLA2 isoenzyme and that DAG could serve as an alternative source for arachidonic acid and seems to sensitize a cytosolic PLA2.
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PMID:Endothelial eicosanoid metabolism and signal transduction during exposure to oxygen radicals injury. 927 14

The staphylococcal alpha-toxin (Hla) is a major virulence factor contributing to Staphylococcus aureus pathogenesis. To elucidate the conditions influencing hla expression, the determinant was fused to lacZ, the reporter gene coding for beta-galactosidase. The hla::lacZ fusion was integrated into the chromosome of the wild-type S. aureus strain Wood 46, leading to the variant Wood 46-3. Alpha-toxin expression was found to be dependent on temperature, showing a maximum at 42 degrees C. Furthermore, the indicator strain showed a growth phase-dependent hla regulation which was influenced by temperature. At 37 degrees C, induction of hla::lacZ expression occurred in the late exponential phase of growth, whereas at 42 degrees C, a strong induction was observed as early as the mid-exponential phase. These observations were verified by Northern blot analysis of hla mRNA and by Western blot (immunoblot) analysis of culture supernatants of strain Wood 46. It was additionally found that the induction of hla transcription at 42 degrees C was not coupled with higher concentrations of agr RNAIII, the effector molecule of the global regulator agr. Furthermore, expression of the alpha-toxin was repressed at a high osmolarity. It was also shown that oxygen is essential for hla expression and that cultivation of the S. aureus strain Wood 46-3 on solid medium and in the presence of carbon dioxide stimulated hla transcriptional activity.
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PMID:Analysis of expression of the alpha-toxin gene (hla) of Staphylococcus aureus by using a chromosomally encoded hla::lacZ gene fusion. 928 26

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O2.-) generation and O2 consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O2.- generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca2/]i and the formation of inositol trisphosphate (IP3) were significantly inhibited by norathyriol (30 microM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 microM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O2.-, however, the inhibition of O2.- generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.
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PMID:Examination of the inhibitory effect of norathyriol in formylmethionyl-leucyl-phenylalanine-induced respiratory burst in rat neutrophils. 935 47

Endothelial cells of the human umbilical blood vessels are widely cultured in an oxygen tension (21%) far above that in which they exist in vivo (3%). This study investigates the effect of the long term culture (ca. 1 month) of human umbilical artery endothelial cells in a reduced oxygen environment (3%: HUAEC3) in comparison to cells grown in a 'normoxic' environment (21%: HUAEC21). Despite reports of altered metabolic pathways and reduced membrane integrity in other cell types, the characteristics of HUAEC3 were found to be similar to those of HUAEC21 with respect to morphology, immunocytochemical profile and in vitro growth rates. Cellular glutathione was maintained in these cells although ATP levels in HUAEC3 were found to be significantly lower than those observed in HUAEC21. The phosphoinositide responses of the HUAEC3 to a variety of agonists were also found to be of similar magnitude to those observed in HUAEC21. In addition, the pharmacological characteristics of the phospholipase C-linked histamine H1 and P2y2 (P2U) receptors were not changed by culture of cells in a low oxygen environment.
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PMID:Receptor-stimulated phospholipase C activity in human umbilical artery cultured endothelial cells grown in a low oxygen environment. 939 78

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.
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PMID:Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis. 952 77

Respiratory syncytial virus (RSV) activated the RelA (p65) subunit of nuclear factor kappa B (NF-kappaB) over many hours postinfection. The initial activation coincided with phosphorylation and degradation of IkappaBalpha, the cytoplasmic inhibitor of RelA. During persistent activation of NF-kappaB at later times in infection, syntheses of inhibitors IkappaBalpha as well as IkappaBbeta were restored. However, the resynthesized IkappaBbeta was in an underphosphorylated state, which apparently prevented inhibition of NF-kappaB. Use of specific inhibitors suggested that the pathway leading to the persistent-but not the initial-activation of NF-kappaB involved signaling through protein kinase C (PKC) and reactive oxygen intermediates of nonmitochondrial origin, whereas phospholipase C or D played little or no role. Thus, RSV infection led to the activation of NF-kappaB by a biphasic mechanism: a transient or early activation involving phosphorylation of the inhibitor IkappaB polypeptides, and a persistent or long-term activation requiring PKC and the generation of hypophosphorylated IkappaBbeta. At least a part of the activation was through a novel mechanism in which the viral phosphoprotein P associated with but was not dephosphorylated by protein phosphatase 2A and thus sequestered and inhibited the latter. We postulate that this led to a net increase in the phosphorylation state of signaling proteins that are responsible for RelA activation.
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PMID:Persistent activation of RelA by respiratory syncytial virus involves protein kinase C, underphosphorylated IkappaBbeta, and sequestration of protein phosphatase 2A by the viral phosphoprotein. 962 Oct 19


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