Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sphingomyelin (SM) biosynthesis is believed to occur in the early Golgi apparatus, plasma membrane and recycling endosomes. In the present study, the localization of the SM synthesis that follows its hydrolysis upon activation of the SM signal-transduction pathway was investigated in human skin fibroblasts treated with tumour necrosis factor (TNF) alpha. After TNFalpha-induced degradation, the intracellular SM levels returned to baseline levels within 30-60 min in cells treated at 37 degrees C. Pretreatment or co-incubation of cells with bacterial sphingomyelinase or phospholipase C, decreasing the SM and phosphatidylcholine content in the external leaflet of the plasma membrane respectively, did not inhibit SM resynthesis. However, SM resynthesis was not observed when TNFalpha-treated cells were continuously exposed to exogenous sphingomyelinase, suggesting that under these particular conditions the resynthesized SM becomes accessible to the enzyme. Furthermore, whereas inhibition of vesicular traffic/endocytosis at 4 degrees C blocked exoplasmic SM resynthesis, it did not alter SM resynthesis in TNFalpha-treated fibroblasts, negating the role of endosomes and the Golgi apparatus. This was further evidenced by the finding that after SM resynthesis, TNFalpha was again able to promote SM turnover, even at 4 degrees C. In addition, when the exoplasmic leaflet SM was hydrolysed by treating fibroblasts with bacterial sphingomyelinase, resynthesis of SM occurred at 37 degrees C much more slowly than after TNFalpha treatment. These findings support strongly the conclusion that the SM, which is resynthesized after TNFalpha-induced hydrolysis, resides in the cytosolic leaflet of the plasma membrane, and that the process involved in this resynthesis displays characteristics different from those of the previously described SM synthases.
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PMID:The tumour necrosis factor-sensitive pool of sphingomyelin is resynthesized in a distinct compartment of the plasma membrane. 963 67

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.
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PMID:Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells. 1501 14

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.
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PMID:The role of intranuclear lipids. 1551 99

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus has been assayed on large and small unilamellar vesicles consisting of PI, either pure or in mixtures with other lipids. Vesicle diameter (in the 50-300 nm range) influences PI-PLC activity, enzyme rates increasing with decreasing curvature radii. With sonicated unilamellar vesicles of pure PI, two apparent K(s) values are observed, one in the 0-2 mM concentration range and the other in the 2-12 mM concentration range. The latter ( approximately 4.2 mM) corresponds to previously published values, while the low-concentration K(s) is on the same order of magnitude as the single apparent K(m) value found with large unilamellar liposomes ( approximately 0.30 mM). PI-PLC appears to be very sensitive to bilayer composition. Certain nonsubstrate lipids, e.g., galactosylceramide or cholesterol, inhibit PI-PLC in a dose-dependent way, at least up to 33 mol % in the bilayers, under conditions with a constant PI concentration. Simultaneous measurements of enzyme activity, interfacial enzyme binding, and fluorescence of different probes, on a variety of bilayer compositions, reveal that both the level of enzyme binding and activity decrease with increasing lipid order, as measured by the fluorescence polarization of the hydrophobic probe diphenylhexatriene. In contrast, no correlation is found for enzyme activity with fluorescence changes of probes, e.g., laurdan, that report on phenomena occurring mainly at the lipid-water interface. Sphingomyelin has a dual effect. Up to 40 mol %, it increases PI-PLC activity, with little effect on bilayer molecular order. At higher proportions, the increased lipid chain order causes a decrease in enzyme activity. The same effects are observed for distearoylphosphatidylcholine when added to PI bilayers. These results support the "two-stage model" for binding of PI-PLC to lipid bilayers, and underline the significance of the enzyme partial penetration into the membrane hydrophobic matrix for its catalytic activity.
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PMID:Modulation of PI-specific phospholipase C by membrane curvature and molecular order. 1611 96

Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potentials in cosmetic and pharmaceutical industries such as in hair and skin care products. Chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient production methods are of great interest. Present study was the first attempt to perform a systematic study on the production of ceramide through enzymatic hydrolysis of sphingomyelin. Sphingomyelin hydrolysis proved to be more efficient in two-phase (water:organic solvent) system than in one-phase (water-saturated organic solvent) system. Among the screened phospholipase C, the Clostridium perfringens enzyme had the highest sphingomyelin conversion rate, with very small temperature dependence. Addition of ethanol to the system markedly enhanced the rate of ceramide formation, and a mixture of ethylacetate:hexane (50:50) was the best organic solvent tested. Other factors such as (NH(4))(2)SO(4), NaCl and CaCl(2) were also tested but excluded for further consideration. On the basis of the initial experiments, the reaction system was optimized using response surface methodology including five factors (enzyme amount, water amount, ethanol amount, reaction time and the hexane ratio of organic solvent). Water content and enzyme amount was shown to have the most significant influence on the hydrolysis reaction in the fitted quadratic model. The efficiency of sphingomyelin hydrolysis was dramatically improved through system evaluation and optimization, with the optimal conditions at 75 min reaction time, 3 Uml(-1) enzyme amount, 6% water amount, 1.8% ethanol amount and 46% hexane in ethylacetate.
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PMID:Enzymatic production of ceramide from sphingomyelin. 1633 3

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.
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PMID:Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol. 1958 91

Sphingomyelin (SM), a major sphingolipid in the lipid raft microdomains of the cell membrane, is synthesized by plasma membrane-bound sphingomyelin synthase 2 (SMS2). SMS2 is required for the maintenance of plasma membrane microdomain fluidity and receptor-mediated responses to inflammation in macrophages. However, the exact mechanism of SMS2 activation in endothelial barrier disruption and lung injury is not fully understood. To define the role of SMS activation in lung injury, we hypothesized that the inhibition of SM synthesis may provide protection against acute lung injury (ALI) by preserving endothelial barrier function. Using SMS2-silencing RNA (siRNA) treatment in human pulmonary endothelial cells (HPAECs) and tricyclodecan-9-yl-xanthogenate (D609), a competitive inhibitor of SMS, and phosphatidylcholine-specific phospholipase C in a murine model of bacterial LPS injury, we studied the role of sphingomyelin synthesis in ALI. Results show that pretreating mice with D609 significantly attenuated LPS-induced lung injury, as measured by a significant decrease in wet to dry ratio, bronchoalveolar lavage fluid cell and protein counts, and myeloperoxidase activity in lung tissue. Similarly, LPS-induced endothelial barrier disruption was significantly reduced in HPAECs pretreated with D609 or SMS2 siRNA, as demonstrated by an increase in paracellular integrity on an FITC-dextran assay, by the inhibition of LPS-induced stress fibers, and by the formation of cortical actin rings and lamellipodia at the periphery. These results indicate that D609 attenuates LPS-mediated endothelial barrier dysfunction and lung injury in mice through inhibition of SMS, suggesting a novel and essential role of SMS inhibition in modulating endothelial barrier integrity via actin cytoskeletal activation, with a potential therapeutic role in ALI.
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PMID:Role of sphingomyelin synthesis in pulmonary endothelial cell cytoskeletal activation and endotoxin-induced lung injury. 2236 86


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