Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells (original) (raw)
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Oncology Reports, 2011
the plasma membrane lipid composition in AH-130 hepatoma cells was found to change remarkably after polyenylphosphatidylcholine (ppc) treatment. plasma membranes from cells grown in rats treated for 7 days i.v. with 20 mg/kg/ day ppc, when compared to those of control cells, did not show significantly different amounts of cholesterol or phospholipids relative to protein content, but, surprisingly, the individual phospholipid distribution inside the two membrane leaflets changed dramatically. phosphatidylcholine (pc), the major phospholipid in the external membrane leaflet, increased ~47% (p<0.001). By contrast, phosphatidylethanolamine (pe), the most important component of the inner leaflet, decreased nearly 37% (p<0.001), while sphingomyelin (SM) also decreased ~17%, (p=0.1). Tumor cells collected from control rats at the same time interval and observed by scanning electron microscopy, exhibited a spherical shape with numerous and randomly distributed long microvilli, the same morphological and ultrastructural features displayed by the implanted cells. conversely, tumor cells from ppc-treated rats no longer showed the roundish cell profile, and microvilli appeared shortened and enlarged, with the formation of surface blebs. Transmission electron microscopy observations confirmed the morphological and ultrastructural cell changes, mainly seen as loss of microvilli and intense cytoplasmic vacuolization. taken together, these results indicate that the new phospholipid class distribution in the plasma membrane leaflets, modifying tumor cell viable structures, produced heavy cell damage and in many cases brought about complete cellular disintegration.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
Rat platelets have been hydrogenated in the presence of colloidal palladium adsorbed on the surface of the non water-soluble polymer polyvinylpolypyrrolidone. This non-permeating catalyst restricts hydrogenation of the fatty acyl double bonds of phospholipids only in the outer half of the plasma membrane. The pattern of hydrogenation of the molecular species present on the external side of the membrane is determined using desorption-chemical soft ionization-mass spectrometry (DCI-MS) before and after cell activation by the calcium ionophore A23187. The accessibility to the catalyst of the polyunsatured molecular species within each phospholipid class is compared for resting and activated cells. The abundance of polyunsaturated species of phosphatidyl-ethanolamine and -serine in the inner half of the resting biomembrane is confirmed in rat platelets. Phosphatidylcholine is especially rich in disaturated species in this membrane. The induced exposure of the polyunsaturated species of diacyland ether-phosphatidylethanolamine, and of phosphatidylserine on the external side of the membrane appears after activation by the calcium ionophore. A detailed quantitative analysis within a phospholipid class shows an unequal scrambling for diacyl-, alkyl-, alkenyl-phosphatidylethanolamine, and a variable involvement in the transmembrane redistribution following cell activation of the various molecular species as a function of the at3,1 moities.
Turnover of phosphocholine and phosphoethanolamine in ether-phospholipids of krebs II ascite cells
Lipids, 1985
Krebs II ascite ceils suspended in Eagle medium were incubated at 37 C for up to 6 hr in the presence of [3H] glycerol or [32p] orthophosphate. After extraction, their lipids were treated with guinea pig phospholipase A, under conditions where all diacyl-phospholipids (diacyl-PL) became hydrolyzed with 55% recovery of lyso-PL. Using a bidimensional thin layer chromatography (TLC) involving exposure to HCI fumes between the two runs, it then became possible to determine at once the specific radioactivity of the three subclasses (diacyl-, alkylacyl-and alkenylacyl-) present in choline glycerophospholipids (CGP) and ethanolamineglycerophospholipids (EGP). Compared to diacyl-PL, a lower de novo synthesis of ether subclasses was evidenced in both CGP and EGP by [3H] glycerol incorporation. Although the same profile was obtained for CGP with [~'P] orthophosphate, the three EGP subclasses displayed in this case the same specific radioactivity.
Drug-induced surface membrane phospholipid composition in murine fibroblasts
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
The effects of drugs on phospholipid composition of cell surface membranes are not well understood at this time. The effects of membrane-active drugs and membrane depolarization on the phospholipid composition were determined in murine LM fibroblasts. Receptor-aggregating drugs such as concanavalin A and cytoskeleton<iisrupting agents such as colchicine, vinblastine, and cytochalasin B decreased phosphatidylserine content of the plasma membrane from 5.4 +-1.5% to as low as 1.4-+ 0.2%. In addition, concanavalin A and colchicine increased the phosphatidylglycerol content from 6.9-+ 1.6% to 13.1 + 0.7% and 10.6-+ 1.7%, respectively, while vinblastine and cytochalasin B had no effect. Pentobarbital decreased the content of phosphatidylinositol + phosphatidylserine and of phosphatidylglycerol almost 2-fold. Propranolol, ethanol, and depolarization with 120 mM KC1 had small or no effects on plasma membrane phospholipid composition. None of the above drugs or treatments significantly altered the asymmetric distribution of phosphatidylethanolamine across the LM cell plasma membrane under the conditions tested. In addition, energy inhibitors that deplete the proton-motive force of the cell (NAN3 and KCN) and inhibitors of ATP synthesis such as NaAsO4 did not affect the asymmetric distribution of phosphatidylethanolamine. It is concluded that the mechanism of action of membrane-active drugs such as concanavalin A, vinblastine, colchicine and pentobarbital may involve alterations in plasma membrane composition. It also appears that microfilaments, microtubules, ~-adrenergic receptors, membrane fluidity, and membrane potential are not critical for the regulation of the asymmetric distribution of membrane phosphatidylethanolamine.
Altered phospholipid composition affects endocytosis in cultured LM fibroblasts
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
The phospholipid polar head group composition of LM fibroblast membranes was altered by growing the cells in a chemically defined, serum-free medium containing choline, N,N'-dimethylethanolamine, N-monomethyiethanolamine, or ethanolamine. The cells incorporated these bases into their membrane phospholipid such that 29-40% of the total plasma membrane phospholipids contained these polar head groups. Alteration of the phospholipid composition correlated with a depression of polystyrene bead phagocytosis by 36, 55 and 85% when the cells had been supplemented with N,N'-dimethylethanolamine, N-monoethylethanolamine, or ethanolamine, respectively. Pinocytotic uptake of horseradish peroxidase was depressed 44, 39, and 32%, respectively. The phagosomal membrane phospholipid composition qualitatively resembled that of the primary plasma membrane from which it was derived. However, enrichment of phosphatidylcholine, and other quantitative differences were noted in the phagosomal membranes as compared to the parent primary plasma membrane. Approx. 50% of the phagosomal membrane's phosphatidylethanolamine was accessible to the chemical labelling reagent trinitrobenzenesulfonate at 4°C. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane did not appear to be altered by base analogues except in the case of phagosomes from cells supplemented with ethanolamine. The data were consistend with a nonrandom site for endocytosis with regard to phospholipid composition.
Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism
Journal of Cellular Biochemistry, 1989
The biosynthesis of platelet-activating factor (PAF-acether or 1 -0-alkyl-2-acetylsn-glycerc-3-phosphoholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A similar study was undertaken on the tumor strain Krebs-I1 cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A, in providing lyso-PAF to the acetyltransferase to form PAF-acether.
Biochimica et biophysica acta, 1984
A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by ph...
Studies on ether phospholipids
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1984
A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A, and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phosphoIipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42°C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A, attack. Phospholipids are then separated by a bidimensio~~ thin-layer chromato~aphy involving the exposure of the plates to HCI fumes between the two runs, in order to hydrolyse piasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phosphoiipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.