Altered phospholipid composition affects endocytosis in cultured LM fibroblasts (original) (raw)
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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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
- Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A 2 and Staphylococcus aureus sphingomyelinase C and on phospholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylchoUne, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyi species by guinea pig pancreas phospholipase A t and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since l-alkyl-2-acyl-sn-glyceroi-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl-and alkenylacyI-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for l-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyi-GPC).
Biochimica et biophysica acta, 2015
Ethanolamine plasmalogens constitute a group of ether glycerophospholipids that, due to their unique biophysical and biochemical properties, are essential components of mammalian cellular membranes. Their importance is emphasized by the consequences of defects in plasmalogen biosynthesis, which in humans cause the fatal disease rhizomelic chondrodysplasia punctata (RCDP). In the present lipidomic study, we used fibroblasts derived from RCDP patients, as well as brain tissue from plasmalogen-deficient mice, to examine the compensatory mechanisms of lipid homeostasis in response to plasmalogen deficiency. Our results show that phosphatidylethanolamine (PE), a diacyl glycerophospholipid, which like ethanolamine plasmalogens carries the head group ethanolamine, is the main player in the adaptation to plasmalogen insufficiency. PE levels were tightly adjusted to the amount of ethanolamine plasmalogens so that their combined levels were kept constant. Similarly, the total amount of polyun...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
at 0-4°C with increasing amounts of fluorescamine. Phospholipids were extracted and the amounts of phosphatidylethanolamine and of its fluorescamine derivative were determined. (1). The plasma membrane of intact Friend cells appeared to be permeable to fluorescamine in a concentration-dependent way. (2). Three pools of phosphatidylethanolamine could be detected as the fluorescamine concentration was raised. The two first pools were ascribed to the outer monolayer (16-17% of the total cellular phosphatidylethanolamine) and inner (17-18%) monolayer of the plasma membrane, respectively, indicating an essentially symmetrical distribution of this phospholipid. The third pool of phosphatidylethanolamine (66%) corresponds to the contribution of intracellular membranes. (3). These data were used in turn, to calculate the relative amount of each phospholipid class present in the plasma membrane. The results are in perfect agreement with those obtained by an independent method involving the use of sphingomyelinase C (Rawyler, A., Roelofsen, B., Op den Kamp, J.A.F. and Van Deenen, L.L.M. (1983) Biochim. Biophys. . The present method is discussed in terms of its applicability for the localization of phosphatidylethanolamine in eukaryotic cells.
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.
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1983
Previous studies on neuroblastoma cells in culture showed that the presence of partially purified rat liver phospholipid-transfer protein had a marked differential effect on the uptake and apparent subcellular distribution of radioactively labeled sphingomyelin and phosphatidylcholine (PC) added to the medium as mixed phospholipid (PC/sphingomyelin) liposomes. To determine the effect of phospholipid-transfer protein and exogenous phospholipids on the turnover and subcellular distribution of endogenous phospholipids, neuroblastoma cells were preincubated for 48 h in the presence of [methyl-3Hlcholine and washed. Aliquots of prelabeled cells were reincubated immediately in medium containing phospholipid-transfer protein mixed phospholipid liposomes, cytochalasin B and 2-deoxyglucose for 45 min at 37°C; additional aliquots were chased first for 2 or 18 h with unlabeled choline before reincubation. The extent of labeled phospholipid degradation and accumulation in the medium, and the subcellular distribution of cell-associated labeled choline-containing phospholipids were determined. During incubation with phospholipid-transfer protein and mixed phospholipid liposomes, 25-35s of the cell-associated radioactive label from prelabeled cells, chased or unchased, was lost to the medium in 45 min. Over 50% of the label appearing in the medium was in water-soluble phospholipid degradation products. The loss of cell-associated label into the medium from unchased cells was stimulated significantly by phospholipid-transfer protein; however, prelabeled cells which had been chased for 18 h with unlabeled choline were unaffected by the presence of transfer protein. Endogenously synthesized radioactively labeled PC and sphingomyelin were distributed throughout all subcellular membranes, but least of all in the crude mitochondrial membrane fraction. Analysis of the subcellular distribution of cell-associated label remaining in chased or unchased cells after 45 min incubation with PC/sphingomyelin liposomes showed proportionate losses from all membrane fractions, except the crude mitochondrial fraction, which showed relative retention of labeled phospholipid. Phospholipid-transfer protein had no effect. The results are in distinct contrast to observations on the turnover, metabolism and subcellular distribution of labeled exogenous phospholipids under the same conditions, indicating that exogenous phospholipids do not intermix freely with any quantitatively major pool of endogenous phospholipid.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.