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. We examined whether or not caffeine caused an endothelium-dependent contraction (EDC) in canine mesenteric artery and whether the endothelium-dependent contracting factors (EDCF) were arachidonic acid metabolites. 2. Caffeine (1, 3 and 10 mM) caused a transient contraction in endothelium-intact arterial strips. Removal of the endothelium significantly attenuated the caffeine (1 and 3 mM)-induced contraction. 3. Caffeine (1 mM)-induced EDC was not affected by quinacrine and manoalide (phospholipase A2 inhibitors), indomethacin and aspirin (cyclo-oxygenase inhibitors), ONO-3078 and S-1452 (thromboxane A2 antagonists) or AA-861 and TMK-777 (lipoxygenase inhibitors). 4. Caffeine (1 mM)-induced EDC was also unaffected by 50-235 (an endothelin A receptor antagonist). In addition, catalase combined treatment with superoxide dismutase, or allopurinol (antioxidant) did not affect the EDC. 5. Gro-PIP and NCDC (phospholipase C inhibitors) did not affect the caffeine-induced EDC. However, wortmannin (a phospholipase D inhibitor) and staurosporine (a protein kinase C inhibitor) attenuated the caffeine-induced EDC. 6. The present experiments demonstrate that caffeine causes an EDC in canine mesenteric artery and suggest that the EDCF mediating this response is probably not arachidonic acid metabolites, endothelin or superoxide. Instead, caffeine-induced EDC may be due to activation of the phospholipase D pathway.
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PMID:An endothelium-dependent contraction in canine mesenteric artery caused by caffeine. 800 88

Phosphatidylinositol 4,5-bisphosphate, a substrate in the signal transduction pathway involving phospholipase C, is synthesized in cell membranes by phosphorylation of phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI-4-P). Incubation with [gamma-32P]adenosine triphosphate of membranes isolated from primary glial cells in culture and from an immortalized astrocyte cell line (DITNC) and subsequent separation of the lipids by high-performance thin-layer chromatography revealed a number of labeled lipid bands. Further analysis of the deacylated products by high-performance liquid chromatography indicated the presence of two PI-4-P bands and a band corresponding to lysoPIP (PIP, PI phosphate). The two PI-4-P bands were more prominent in the samples from astrocyte cell membranes than from synaptic plasma membranes and plasma membranes. Analysis of the fatty acid composition of PI by gas-liquid chromatography revealed that both 20:3n-9 and 20:4n-6 were present in PI of cultured cell membranes, whereas the brain membranes contained exclusively 20:4n-6. The two PI-4-P bands in these membranes can be attributed to the presence of different molecular species of PI. Assignment of the fraction corresponding to lysoPIP was consistent with the observation that an increase in label in this lipid band occurred upon incubation of DITNC cell supernatant with lysoPI (1-stearoyl PI). This suggests that endogenous lysoPI present in the cell membranes can be phosphorylated to form lysoPIP.
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PMID:Phosphorylation of lipids in rat primary glial cells and immortalized astrocytes (DITNC). 809 58

Two forms (I and II) of phosphoinositide-specific phospholipase C (PLC) were purified from the cytosol of bovine iris sphincter by sequential chromatography on DEAE-Sepharose, EAH-Sepharose, heparin-Sepharose, Sephacryl S-200 gel filtration and Mono Q HR columns. The final step resulted in specific activities of PLC-I and PLC-II of 4.3 and 5.9 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein, which represented up to 295-fold purification compared with that of the starting supernatant. The purified enzymes were further investigated for the presence of isoenzymes and characterized for molecular mass, substrate specificity, pH, Ca2+ requirements and kinetic parameters. Using monoclonal antibodies, PLC-I was identified as PLC-delta 1. The apparent molecular mass of PLC-I as determined by SDS/PAGE and gel filtration was 85 kDa. PLC-II contained an apparently invisible protein band that reacted with the antibody against PLC-gamma 1, and a major 109 kDa protein band that was not recognized by any of the PLC monoclonal antibodies. Further purification of PLC-II by size-exclusion h.p.l.c. resulted in elution of the enzyme activity as a single peak which corresponded to 109 kDa position. Again, this PLC activity was not recognized by any of the PLC monoclonal antibodies. However, the 109 kDa protein activity was recognized by a polyclonal antibody raised against a rat PLC-gamma 1 fragment (amino acids 1272-1287), thus suggesting that this protein is a proteolytic product of PLC-gamma 1. PLC-delta 1 and PLC-gamma 1 were identified in the supernatant fraction and PLC-beta 1 in the membrane fraction of the iris sphincter. Although immunologically different, the catalytic properties of PLC-I and PLC-II were quite similar. The Vmax and Km values for phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis were three to five times greater than those for PI hydrolysis. Both forms preferred PIP and PIP2 over PI and both were inactive against phosphatidylcholine. With PIP2 as substrate, the optimal pH values for PLC-I and PLC-II were 6.5 and 7.5 respectively. Unlike PIP2, PI hydrolysis by both forms was dependent on the presence of free Ca2+. The maximal hydrolysis of PI and PIP2 by both forms occurred at 200 and 5 microM Ca2+ respectively. Incubation of the purified enzymes with the catalytic subunit of protein kinase A (PKA) and [gamma-32P]ATP resulted in increased phosphorylation of PLC-I and PLC-II, but it had no inhibitory effect on their enzyme activities.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Purification and characterization of phosphoinositide-specific phospholipase C from bovine iris sphincter smooth muscle. 838 Sep 92

The effect of nitrogen-(N2-)microbubbles on platelets resembles that of common platelet agonists with respect to aggregation and secretion, but is considerably slower and is poorly inhibited by aspirin. This paper reports the effect of microbubbles on platelet phospholipase C activity in gelfiltered human platelets prelabelled with [32P]Pi ([32P]-GFP). The experiments were run in the presence of an ADP scavenging system in order to rule out effects of ADP. Stimulation of [32P]-GFP for 30 min with microbubbles caused a significant reduction in single platelets (p < 0.0004) and a significant increase in 32P-activity in the phosphatidic acid (PA) fraction (p < 0.02). Epinephrine potentiated the microbubble-induced reduction in single platelets (p < 0.05), but did not enhance the amount of 32P in the platelet [32P]PA fraction. The 32P-radioactivity in the PI-fraction increased with time to a similar extent when [32P]-GFP was stirred for 30 min in absence of microbubbles as it did after 30 min of agonist exposure. There were no significant changes in the [32P]PIP and [32P]PIP2 fractions. Aspirin abolished the microbubble-induced increase in 32P-activity in the PA fraction, but had no significant effect on the reduction in single platelets. Aspirin had a small but significant, reducing effect on platelet aggregation induced by a combination of epinephrine and microbubbles (p < 0.05). With epinephrine, however, aspirin did not completely abolish the increase in [32P]-PA. It is concluded that microbubbles alone cause platelets to aggregate by a novel mechanism that operates independent of cyclooxygenase-dependent arachidonic acid metabolites and phospholipase C activation.
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PMID:Microbubble-induced phospholipase C activation does not correlate with platelet aggregation. 838 81

We examined the rate and extent of labeling of total cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI) in canine tracheal smooth muscle in response to maximum levels of two different contractile agonists, carbachol (5.5 microM) and serotonin (5-hydroxytryptamine, 5-HT) (47 microM) and when a second agonist was given during a maximal contraction evoked by the first agonist. Unstimulated tracheal smooth muscle strips were incubated with [3H]-myo-inositol (MI) to label tissue MI without much labeling of inositol phospholipids. With carbachol, there was a 20-fold increase in the [3H]-MI incorporation rate into inositol phospholipids, decreases in PI and PIP2 contents, and increases in phosphatidic acid and diacylglycerol contents. PI and PIP2 specific radioactivities reached plateaus, 0.90 +/- 0.03 and 0.80 +/- 0.04, respectively, compared with [3H]-MI specific radioactivity. 5-HT at 47 microM evoked smaller changes including force development, [3H]-MI incorporation rate and lipid mass changes. However, the plateau of PI and PIP2 labeling reached levels similar to that determined during carbachol-evoked force, 0.90 +/- 0.06 and 0.82 +/- 0.04, respectively. Carbachol (55 microM) addition after incubation with 5-HT did not significantly alter the plateau levels of the specific radioactivities of PI or PIP2, although force and lipid mass changes were significantly changed. We conclude that 5-HT and muscarinic receptor coupling mechanisms utilize the same pool of PIP2 as a substrate for phospholipase C activation and the same PI pool for conversion to PIP and PIP2.
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PMID:Common phosphatidylinositol 4,5-bisphosphate pools are involved in carbachol and serotonin activation of tracheal smooth muscle. 839 64

The purpose of these studies was to examine the sensitivity of the PIP 2-PLC-transducing pathway (GPLC) and its relationship to the respiratory burst in human polymorphonuclear leukocytes (PMN) stimulated by IL-8, TNF-alpha, or IL-1 beta during sequential changes in buffer oxygen tension from normoxia (pO2 = 180-200 mm Hg), to hypoxia (pO2 < 30 mm Hg) and then reoxygenation (pO2 > 140 mm Hg). Our specific hypothesis was that altered oxygen tensions would regulate the G PLC pathway in human PMN. G PLC activity was assayed by investigating phospholipase C activity by measuring inositol phosphates and diacylglycerol (DAG) formation. Respiratory burst activity was assayed as O 2 production and NADPH oxidase activation in intact PMN and in a cell-free system, respectively, and correlated separately to both early and late DAG production. At 1 min, DAG formation during normoxia was decreased by IL-8 plus fibronectin while hypoxia had no regulatory effect on control of DAG formation by any of the cytokines. In contrast to early DAG formation, hypoxia significantly downregulated late DAG formation induced by buffer without fibronectin, IL-8 plus fibronectin, and IL-1 beta with or without fibronectin. Hypoxia/reoxygenation in and of itself significantly increased DAG formation vs levels seen in the presence or absence of IL-8, TNF-alpha, or IL-1 beta with or without fibronectin. Changes in early DAG production during the alterations in oxygen tension correlated best with corresponding changes in O 2 production in intact cells, whereas late DAG production correlated best with NADPH oxidase activation assayed in the cell-free system. Thus, changes in oxygen tension can directly modulate the extent of the PMN response to stimulation by IL-8, TNF-alpha, or IL-1 beta and the G PLC-receptor pathway is particularly regulated by physiologically relevant periods of hypoxia/reoxygenation.
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PMID:Altered oxygen tension modulates cytokine-induced signal transduction in polymorphonuclear leukocytes: regulation of the G PLC pathway. 860 6

Extracellular matrix (ECM) molecules, such as fibronectin (FN), regulate fibroblast sensitivity to soluble growth factors, in part, by controlling cellular levels of phosphatidylinositol bis-phosphate (PIP2), the substrate for phospholipase C-gamma (McNamee et al., 1993, J. Cell Biol. 121, 673-678). In the present study, we extended these investigations by exploring whether cells of the vascular wall also exhibit this response and analyzing the mechanism by which adhesion to ECM regulates intracellular PIP2 mass. Capillary endothelial cells, pulmonary vascular smooth muscle cells, and C3H 101/2 fibroblasts were all found to exhibit a similar two- to threefold increase in PIP2 mass within 3 h after binding to dishes coated with FN. Furthermore, similar effects were observed using dishes coated with a variety of different ECM molecules, including collagen types I and IV as well as a synthetic RGD-containing peptide. An increase in PIP2 mass also was produced when suspended cells bound to microbeads (4.5 micron diameter; coated with RGD-peptide or anti-integrin beta 1 antibody) that induce local integrin clustering and focal adhesion formation, independently of cell spreading. In contrast, neither binding of soluble FN nor binding of microbeads coated with ligands for other transmembrane surface receptors (e.g., acetylated low-density lipoprotein, antibodies against heparan sulfate) had any effect on PIP2 mass. While these results suggest that integrin clustering stimulates PIP2 synthesis, no change in total cellular or cytoskeletal-associated phosphatidylinositol-4-phosphate kinase (PIP kinase) activity could be detected when cells bound to immobilized integrin ligands. However, when focal adhesion complexes were isolated from these cells using a magnetic procedure (G. Plopper and D. E. Ingber, 1993, Biochem. Biophys. Res. Commun. 193, 571-578), this subfraction of the cytoskeleton was found to be enriched for PIP kinase activity by more than twofold relative to the whole cytoskeleton. These data suggest that ECM binding may increase PIP2 mass in vascular cells by clustering cell surface integrin receptors and activating cytoskeletal-associated PIP kinases locally within the focal adhesion complex.
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PMID:Integrin-dependent control of inositol lipid synthesis in vascular endothelial cells and smooth muscle cells. 861 75

The endogenous cyclic adenosine monophosphate (AMP) antagonist, cyclic PIP, has been identified as a prostaglandylinositol cyclic phosphate. It inhibits protein kinase A 100% and activates protein serine phosphatase about sevenfold. It is biosynthesized by an enzyme of the plasma membrane when the assay mixture contains adenosine triphosphate (ATP), Mg2+, prostaglandin E and a novel inositol polyphosphate, which cannot be substituted by commercially available inositol phosphates. This novel inositol polyphosphate is a very labile compound. On anion exchange chromatography it elutes in the range of ATP, which may indicate the presence of three phosphate groups. It adsorbs on charcoal, which suggests the presence of a hydrophobic component, possibly a guanosine. Pyrophosphates obtained from inositol 1,4- and inositol 2,4-bisphosphate are accepted by cyclic PIP synthetase for the synthesis of cyclic PIP. The biosynthesis is characterized by enzyme kinetic parameters like dependence on time, enzyme and substrate concentration. The pH optimum of the enzyme is in the range 7.5-8. The enzyme functions optimally with prostaglandin E and poorly with prostaglandin A as the substrate. The presence of fluoride in the assay causes a three- to fourfold increase in cyclic PIP synthesis, which may be correlated with activation via G proteins. These data support previous reports on the chemical structure and action of cyclic PIP. With respect to the possible isomers of cyclic PIP, these indicate that it is most likely the C4-hydroxyl group of the inositol which binds the C15-hydroxyl group of prostaglandin E. A model of hormone-stimulated synthesis of cyclic PIP is proposed: phospholipase A2 and phospholipase C, activated by G proteins upon alpha-adrenergic stimulation, liberate either unsaturated fatty acids or inositol phosphates, which are transformed to prostaglandins and to novel inositol polyphosphate with an energy-rich bond. The cyclic PIP synthetase combines these two substrates to cyclic PIP.
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PMID:Biosynthesis of the endogenous cyclic adenosine monophosphate (AMP) antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), from prostaglandin E and activated inositol polyphosphate in rat liver plasma membranes. 887 Aug 15

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [32P]i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol/1011 cells at 10-5 M forskolin) and [32P]phosphatidylinositol 4-phosphate (Delta[32P]PIP) (30% at 10-7-10-6 M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [32P]PA, 'overshoots' in [32P]PIP and [32P]PIP2 ([32P]phosphatidylinositol 4,5-bisphosphate), and the increase in [32P]PI and secretion of ADP+ATP were abolished by forskolin (10-7 M). Forskolin stimulated total [32P]Pi uptake in resting platelets (48%), increased 32P incorporation into PIP (110%), and inhibited 32P incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the forskolin-induced stimulation of [32P]Pi uptake. The changes in radioactive PA, PIP and PIP2 are regarded as being proportional with their masses in the prelabeled platelets, while the increase in PI (phosphatidylinositol) is regarded as a change in specific radioactivity, and hence in its synthesis. The results suggest that cAMP elevation inhibits the flux in the polyphosphoinositide cycle through both inhibition of PIP 5-kinase and PI synthesis. The inverse relation between forskolin-produced DeltaPIP and [32P]PA production suggests that the PLC reaction is inhibited by elevated cAMP through reduction of substrate (PIP2) resynthesis, and not by inhibition of the PLC enzyme.
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PMID:Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets. 979 33

Recent evidence supporting a role for phosphoinositides in the endocytosis of phospholipase C-coupled receptors has prompted an investigation of whether there exists a similar requirement for the internalization of adenylyl cyclase-linked receptors. When 1321N1 astrocytoma cells, which possess both muscarinic cholinergic receptors (mAChRs) that couple to phospholipase C and beta-adrenergic receptors (beta(2)-ARs) linked to adenylyl cyclase, were pretreated with wortmannin (WT) at a concentration known to inhibit phosphatidylinositol 4-kinase activity, the labeling of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP(2). As previously demonstrated for SH-SY5Y neuroblastoma, inclusion of WT inhibited the endocytosis of mAChRs in 1321N1 cells by >85%. In contrast, the internalization of beta(2)-ARs was only partially ( approximately 30%) prevented. However, when the concentration of PIP(2) was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of beta(2)-ARs was substantially inhibited (>70%). Lower concentrations of WT (100 nM) that were sufficient to fully inhibit phosphatidylinositol 3-kinase activity had no effect on either phosphoinositide synthesis or receptor endocytosis. The results indicate that the agonist-induced endocytosis of an adenylyl cyclase-linked receptor such as the beta(2)-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP(2).
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PMID:Inhibition of beta(2)-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate. 1041 68


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