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)

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.
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PMID:Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. 130 23

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.
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PMID:Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ. 130 41

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.
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PMID:Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. 133 83

1. Cultured aortic endothelial cells of the pig respond to the endothelium-derived relaxing factor (EDRF) they release with an increase in cyclic GMP content. This response is inhibited by haemoglobin or by L-NG-monomethyl-arginine (L-NMMA), and has been used to investigate the effects of phorbol esters on EDRF release. 2. Pretreatment with phorbol-12,13-dibutyrate (PDB) but not the inactive 4 alpha-phorbol-12,13,-didecanoate (PDD), inhibited increases in cyclic GMP induced by substance P (10(-8) M) in a time and concentration-dependent manner. PDB did not affect basal cyclic GMP levels. 3. PDB (3 x 10(-7) M), but not PDD (3 x 10(-7) M), also inhibited ATP (10(-5) M)-induced increases in cyclic GMP, but did not affect those induced by bradykinin (10(-7) M). 4. Increases in cyclic GMP induced by low (10(-7) M) but not high (10(-6) M) concentrations of the calcium ionophore A23187 were inhibited by PDB (3 x 10(-7) M). This inhibitory effect was due to enhanced destruction of EDRF by superoxide anions rather than inhibition of EDRF release, as the inhibition was abolished in the presence of superoxide dismutase (SOD, 30 mu ml-1) and catalase (CAT, 100 mu ml-1). 5. SOD and CAT did not affect the inhibitory action of PDB on substance P or ATP-induced increases in cyclic GMP. 6. Increases in endothelial cell cyclic GMP content induced by sodium nitroprusside (10(-5) M) were unaffected by PDB pretreatment. 7. The inhibitory effects of PDB are probably a result of an action of protein kinase C on the steps between receptor occupation and phospholipase C activation.
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PMID:Release of endothelium-derived relaxing factor from pig cultured aortic endothelial cells, as assessed by changes in endothelial cell cyclic GMP content, is inhibited by a phorbol ester. 169 49

Porcine or bovine endothelial cells cultured on microcarrier beads, packed into adapted chromatographic columns, perfused with Krebs' buffer and activated with appropriate stimuli (e.g. bradykinin, ADP or phospholipase C) release EDRF and prostacyclin into the perfusing fluid. In the effluent EDRF and prostacyclin might be bio-assayed using the Vane's superfusion cascade (rabbit aortic strips and bovine coronary artery strips, respectively) against nitroglycerine (GTN) and synthetic prostacyclin standards. Prostacyclin might be also quantified as 6-keto-PGF1 alpha by RIA. A spatial separation of the generator (endothelial cells) from the effector (vascular smooth muscle) has allowed to prove that EDRF is nitric oxide, that its activity is inhibited by superoxide anions and by chemicals which act via free radicals, finally, that the release of EDRF and prostacyclin is coupled by a receptor-mediated activation of phospholipase C. Although so successful, the above technique suffers from its essentials, i.e. from using cultured cells instead of fresh intact endothelial cells. Cultured endothelial cells are not responsive to many receptor agonists including acetylcholine, substance P and 5-hydroxytryptamine. Unlike fresh intact endothelial preparations the cultured cells which are perfused with Krebs' buffer generate superoxide anions at such concentrations that it might be obligatory infusing superoxide dismutase in order to detect EDRF. Nonetheless, a couple of data obtained with the cultured endothelial cells have been reproduced in the fresh cell preparations, e.g. release of EDRF by ADP and ATP, a coupled release of EDRF and prostacyclin by phospholipase C or a paradoxical augmentation of the sodium-nitroprusside-induced vasorelaxation by methylene blue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endothelium-derived relaxing factor (EDRF) from cultured and fresh endothelial cells. 247 Mar 61

Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F1 alpha. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. We suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.
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PMID:Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled. 283 51

Exposure of isolated SENCAR mouse epidermal cells to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) in vitro resulted in the production of oxidant species detected as chemiluminescence. This oxidant response can be inhibited by superoxide dismutase and copper complexes but not catalase or scavengers of hydroxyl radical or singlet oxygen, suggesting that the oxidant is superoxide anion. Inhibitors of various parts of the arachidonate cascade affect the TPA-induced oxidant response in a manner that corresponds to their effects on in vivo tumor promotion experiments. Agents that inhibit lipoxygenase activity, i.e. nordihydroguaiaretic acid, benoxaprofen, but not agents that are cyclooxygenase inhibitors, i.e. indomethacin, are effective in suppressing the oxidant response to TPA. Phospholipase C but not phospholipase A2 or D produced an oxidant response kinetically similar to that elicited by TPA. The inhibitors of TPA-induced oxidants inhibited the phospholipase C response to the same extent, suggesting that TPA and phospholipase C may produce an oxidant species through a common mechanism, via phospholipid turnover-protein kinase C activation. The relevance of oxidant production to the tumor promotion process is suggested by the ability of exogenous xanthine/xanthine oxidase, a superoxide anion-generating system, to induce ornithine decarboxylase, a characteristic of TPA-treated cells. In addition, oxidant production is significantly lower in cells from the TPA-promotion resistant C57BL/6J mouse. These studies provide further support for a role for reactive oxygens in the tumor promotion process.
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PMID:Reactive oxygen in the tumor promotion stage of skin carcinogenesis. 284 22

Previous work has demonstrated that myocardial ischemia results in a breakdown of the excitation-contraction coupling system of cardiac muscle associated with lysosomal activation. It has been hypothesized that lysosomal activation during the course of myocardial ischemia is mediated by the production of oxygen free radicals. We have tested the hypothesis that myocardial ischemia results in the activation of lysosomal phospholipase C and disruption of calcium transport in sarcoplasmic reticulum (SR) mediated by oxygen free radicals. Three groups of dogs were studied: sham-operated controls (n = 6); normothermic global ischemia of 30-min duration (n = 6); and 30 min of normothermic global ischemia pretreated with intracoronary superoxide dismutase (SOD, 10 micrograms/ml) plus catalase (25 micrograms/ml). In vitro, isolated SR demonstrated a significant depression of calcium uptake rates and Ca2+-stimulated, Mg2+-dependent ATPase activity at both pH 7.0 and 6.4 with the depression at pH 6.4 greater than 7.0. This depression of SR function was significantly inhibited in hearts pretreated with SOD plus catalase. In sham-operated controls, acid-induced dysfunction was associated with substantial loss of phospholipid phosphorus and major changes in phospholipid composition. SR contained an extremely active, ion-independent sphingomyelinase-phospholipase C (SM-PLC) that had maximal activity at pH 4.5-5.0. This SM-PLC was activated when control SR was incubated at acid pH and the specific activity of SM-PLC was decreased 50% in SR isolated from normothermic global ischemia. Activity remained at control levels in hearts pretreated with SOD plus catalase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sarcoplasmic reticulum dysfunction: phospholipid alterations induced by lysosomal phospholipase C. 377 91

The effects of topical application of agents which produce oxygen radicals on cerebral arterioles were studied in anesthetized cats. Xanthine oxidase plus xanthine, which produced superoxide anion radical, hydrogen peroxide, and hydrogen peroxide plus ferrous sulfate, which produced the free hydroxyl radical, induced sustained dilation, reduced responsiveness to the vasoconstrictor effect of hypocapnia, and destructive lesions of the endothelium and of the vascular smooth muscle. Similar effects were produced by arachidonate, 15-HPETE, and PGG2. The effect of arachidonate was inhibited by mannitol, a free hydroxyl radical scavenger, the effect of PGG2 was inhibited by SOD, the effect of 15-HPETE was inhibited by either catalase or SOD. These results suggest that these cerebral vascular abnormalities were produced by a single destructive free radical, probably the hydroxyl free radical, generated via interaction of superoxide and hydrogen peroxide. Cerebral vascular abnormalities similar to those produced by oxygen radicals were also seen after experimental concussive brain injury or after acute hypertension. After brain injury, activation of phospholipase C and increased brain prostaglandin concentration were demonstrated. The vascular effects of brain injury and acute hypertension were inhibited by free radical scavengers. The results suggest that, in these conditions, vascular damage is induced by oxygen radicals generated from arachidonate in association with increased prostaglandin synthesis.
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PMID:Oxygen radicals and vascular damage. 640 99

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


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