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)

A sensitive assay was developed for detection and quantitation of subtle permeability changes in the cytoplasmic membrane of human diploid fibroblasts. Release of the non-metabolizable amino acid [1-14C]alpha-aminoisobutyric acid (AIB; molecular weight (103) from the cytoplasm of prelabeled cells was used as an indicator of toxin-induced membrane damage. An optimal procedure for labeling these cells was designed after varying the conditions with regard to pH, temperature, concentration of AIB, composition of medium, and incubation time. Toxin-induced release of AIB was compared with release of a previously described nucleotide label, [3H]uridine. Melittin from bee venom and the polyene antibiotics filipin and amphotericin B in low concentrations induced a strikingly greater release of AIB than of nucleotide label. The sensitivity of this assay was furthermore demonstrated by treatment with the following bacterial cytolysins: phospholipase C and theta-toxin from Clostridium perfringens, alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus, and streptolysin S from Streptococcus pyogenes. In spite of their different modes of action, all these membrane-active toxins at low concentrations induced a significant release of AIB label. For an equal release of nucleotide label, several times higher concentrations were required.
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PMID:Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. 16 1

Two amphiphilic peptides from hymenopterid insects, melittin and mastoparan, stimulate secretion in a variety of cell types. In PC12 cells, both peptides stimulate calcium influx with melittin some 20-fold more potently than mastoparan. Melittin stimulates both breakdown of phosphoinositides (Pl) by phospholipase C to yield inositol phosphates and hydrolysis of phospholipids by phospholipase A2 to release arachidonic acid (AA). Mastoparan stimulates Pl breakdown, but has no effect on AA release. Maximal stimulation of Pl breakdown occurs at 1 to 2.5 micrograms/ml melittin and 30 micrograms/ml mastoparan, whereas maximal stimulation of AA release occurs at 2 to 5 micrograms/ml melittin. Organic calcium channel blockers (nifedipine, verapamil, diltiazem) have little or no effect on responses to the peptides. The influx of calcium elicited by melittin or mastoparan is completely or nearly completely blocked by inorganic calcium channel blockers (Co++, Mn++, Cd++). Mn++ and Cd++ inhibit melittin-induced Pl breakdown and AA release and mastoparan-induced Pl breakdown. Co++ has no effect on melittin-induced Pl breakdown and potentiates mastoparan-induced Pl breakdown. Pertussis toxin has no effect on the Pl breakdown induced by either peptide. The responses to melittin and mastoparan in PC12 cells are compared to those reported for maitotoxin.
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PMID:Effects of the amphiphilic peptides melittin and mastoparan on calcium influx, phosphoinositide breakdown and arachidonic acid release in rat pheochromocytoma PC12 cells. 130 80

Previous investigations from this laboratory have implicated both phospholipase A2 and phospholipase C in the regulation of human placental lactogen release from human trophoblast. To study further the role of endogenous phospholipase A2 and the relationship between phospholipase A2 activation and phosphoinositide metabolism, we examined hPL and [3H]-inositol release from trophoblast cells in response to agents that stimulate or inhibit the endogenous enzyme. Melittin (0.5-2.0 micrograms/ml) stimulated rapid, dose-dependent, and reversible increases in the release of hPL, prostaglandin E, and [3H]-inositol. Mepacrine (0.1-0.25 mM) inhibited this stimulation. However, mepacrine had no effect on the stimulation of hPL and [3H]-inositol release by exogenous arachidonic acid (AA). These results indicate that the stimulation by melittin of phosphoinositide metabolism and hPL release is mediated by initial activation of phospholipase A2. Furthermore, the results support the possibility that AA, released as a consequence of phospholipase A2 activation, can act as a second messenger linking the two phospholipase pathways.
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PMID:Melittin stimulates phosphoinositide hydrolysis and placental lactogen release: arachidonic acid as a link between phospholipase A2 and phospholipase C signal-transduction pathways. 185 18

The release of eicosanoids and endothelium-derived relaxing factor (EDRF) from endothelial cells is thought to involve a calcium-dependent step. Using cultured bovine aortic endothelial cells as a model system, we have examined the relation between agonist-induced changes in inositol polyphosphates and calcium levels within the endothelial cells and extracellular calcium on EDRF release. In a superfusion-cascade system, EDRF was detected by the relaxation of a rabbit aortic ring without endothelium suspended beneath a column of cultured endothelial cells. Endothelial cell stimulation by bradykinin or melittin induced dose-dependent relaxation of the bioassay ring. In addition, bradykinin and melittin stimulated an increase in intracellular calcium concentration in fura-2 loaded endothelial cells and an increase in inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in cells prelabeled with 3H-myoinositol. Bradykinin stimulation produced transient increases in Ins(1,4,5)P3, fura-2 fluorescence and transient EDRF release. Melittin stimulation induced more prolonged release of EDRF from the endothelial cell column, which was correlated with sustained increases in the fura-2 signal and the level of Ins(1,4,5)P3. Omission of calcium from the cell superfusate attenuated, but did not eliminate, bradykinin-induced EDRF release and the calcium transient, whereas the melittin-induced responses were only slightly attenuated. Endothelial cells clearly demonstrate receptor-activation of phospholipase C and release of sequestered calcium from subcellular sites in response to Ins(1,4,5)P3. These results imply that EDRF release is correlated with increased intracellular calcium levels seen in the absence of extracellular calcium. However, sustained release of EDRF does require influx of extracellular calcium via an undefined mechanism.
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PMID:Endothelium-derived relaxing factor release associated with increased endothelial cell inositol trisphosphate and intracellular calcium. 326 34

The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.
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PMID:The mechanism of stimulation of brain phospholipase C-alpha by myelin basic protein involves specific interactions. 751 86

The ability of the amphiphilic peptides, melittin and mastoparan, to modulate the production of inositol(1,4,5)trisphosphate in cultured plant (Daucus carota L.) cells was investigated. When added to intact cells melittin and mastoparan caused a rapid and dose-dependent increase in inositol(1,4,5)trisphosphate concentrations. In isolated protoplasts, inositol(1,4,5)trisphosphate levels were 12- to 16-fold higher than in the corresponding cells and neither melittin nor mastoparan was able to significantly affect inositol(1,4,5)trisphosphate production. Melittin and mastoparan had a strong inhibitory effect (IC50: 20 microM) on the activity of polyphosphoinositide-specific phospholipase C in purified plasma membranes. These results demonstrate that the plant phosphoinositide system can be activated by amphiphilic peptides in a manner analogous to that observed in specialized mammalian cells but that important functional components are altered, or lost, by the disruption of the intact cell state.
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PMID:Inositol(1,4,5)trisphosphate production in plant cells: stimulation by the venom peptides, melittin and mastoparan. 799 6

Phospholipase A2 (PLA2) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA2-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca(2+)-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 micrograms/ml) significantly stimulated the PLA2 activity and the release of free arachidonic acid (AA) from 1-acyl 2-14C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA2 hyperactivity is CA(2+)-dependent. There was no considerable alteration in the PLA2 activity by stimulation of the activity by tyrosine kinase (with vanadate, H2O2), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF4), thus in Tetrahymena PLA2 activity seems to be independent of these--in Tetrahymena (also functioning)--signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA2 activity is in connection with the phospholipid metabolism of Tetrahymena.
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PMID:PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators. 904 74

Recent studies have suggested the involvement of phospholipase A2 (PLA2) in pancreatic amylase secretion. The present study was designed to investigate the secretory role of arachidonic acid (AA) in carbachol (Cch)-stimulated rat pancreatic acini and its origin. From enzymatic assays, PLA2 and diacylglycerol (DAG) lipase were activated by Cch and respectively inhibited by the PLA2 inhibitors, mepacrine and aristolochic acid, and by the DAG lipase inhibitor, RHC 80267. Melittin-activated PLA2 activity was also inhibited by the PLA2 inhibitors. Cch-stimulated endogenous AA release from pancreatic acini was partially inhibited by 150 microM RHC 80267 and by 150 microM mepacrine or 200 microM aristolochic acid and totally inhibited by a combination of the two enzyme inhibitors. Exogenous AA caused amylase release in a concentration-dependent manner. Eicosatetraynoic acid (a cyclooxygenase and lipoxygenase inhibitor), significantly increased basal and Cch-induced AA release and amylase secretion. RHC 80267 and the PLA2 inhibitors separately and partially suppressed Cch-stimulated amylase secretion, with an additive effect observed when the DAG lipase and the PLA2 inhibitors were combined. A combination of RHC 80267, mepacrine, or aristolochic acid and the phospholipase C (PLC) inhibitor U73122 completely inhibited Cch-stimulated amylase secretion. Finally, the PLA2 activator melittin-stimulated amylase secretion was blocked by the two PLA2 inhibitors. We conclude that exogenous and endogenous AA can induce amylase secretion. Therefore, AA released from either PLC-DAG lipase or PLA2 pathways can be considered an additional and important intracellular mediator of amylase secretion.
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PMID:Endogenous arachidonic acid release and pancreatic amylase secretion. 909 63

We have investigated the possible interaction (cross talk) between the phospholipase A2 (PLA2) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [3H]arachidonic acid ([3H]AA) from [3H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKC alpha and beta immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Melittin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA2 inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [3H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [3H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [3H]AA production was not different from control, whereas SP still displayed [3H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [3H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [3H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of 3H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA2 pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes alpha and beta, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA2 pathway at a level at or beyond PLA2, and this effect is mediated, in part, through PKC alpha and beta species and (for SP) intracellular Ca2+ recruited from internal stores as well as from external sources; and (3) SP also activates PLA2 through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.
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PMID:Cross talk between substance P and melittin-activated cellular signaling pathways in rat lactotroph-enriched cell cultures. 923 37

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.
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PMID:Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin. 1137 Jun 72


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