FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2125973635> ?p ?o ?g. }

• W2125973635 endingPage "33236" @default.
• W2125973635 startingPage "33228" @default.
• W2125973635 abstract "The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established. The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established. The distribution of lipids across the eukaryotic plasma membrane bilayer is asymmetric with the choline phospholipids sphingomyelin and phosphatidylcholine (PC) 1The abbreviations used are: PC, phosphatidylcholine; C6-NBD-, N-6-(7-nitro-2,1,3-benzoxadiazol-4-yl)-aminohexanoyl-; PS, phosphatidylserine; PE, phosphatidylethanolamine; SUV, small unilamellar vesicle; PLA2, phospholipase A2; PCTP, phosphatidylcholine transfer protein (nomenclature of ABC transporters: ABCB1 = MDR1, ABCB4 = MDR3, ABCC1 = MRP1, ABCC7 = CFTR, ABCG2 = BCRP); DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; BSA, bovine serum albumin; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; BFA, brefeldin A. 1The abbreviations used are: PC, phosphatidylcholine; C6-NBD-, N-6-(7-nitro-2,1,3-benzoxadiazol-4-yl)-aminohexanoyl-; PS, phosphatidylserine; PE, phosphatidylethanolamine; SUV, small unilamellar vesicle; PLA2, phospholipase A2; PCTP, phosphatidylcholine transfer protein (nomenclature of ABC transporters: ABCB1 = MDR1, ABCB4 = MDR3, ABCC1 = MRP1, ABCC7 = CFTR, ABCG2 = BCRP); DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; BSA, bovine serum albumin; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; BFA, brefeldin A. at the cell surface and the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) at the inside (1Verkleij A.J. Zwaal R.F. Roelofsen B. Comfurius P. Kastelijn D. van Deenen L.L. Biochim. Biophys. Acta. 1973; 323: 178-193Crossref PubMed Scopus (819) Google Scholar, 2Zwaal R.F. Roelofsen B. Comfurius P. van Deenen L.L. Biochim. Biophys. Acta. 1975; 406: 83-96Crossref PubMed Scopus (279) Google Scholar). PS and PE are continuously translocated from the exoplasmic to the cytoplasmic leaflet of cellular membranes by proteins belonging to a subfamily of P-type ATPases (3Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar, 4Pomorski T. Lombardi R. Riezman H. Devaux P.F. van Meer G. Holthuis J.C. Mol. Biol. Cell. 2003; 14: 1240-1254Crossref PubMed Scopus (293) Google Scholar). Inward translocation of PS is essential to prevent PS cell surface signaling, which induces blood coagulation and serves as a signal for cell-cell recognition, e.g. the removal of apoptotic cells by macrophages. An additional function of lipid translocation has emerged recently. Evidence from mammalian and yeast cells suggests that ATP-dependent inward translocation of phospholipids by the aminophospholipid translocase affects membrane curvature and is a, or the, driving force for the formation of endocytic vesicles at the plasma membrane (4Pomorski T. Lombardi R. Riezman H. Devaux P.F. van Meer G. Holthuis J.C. Mol. Biol. Cell. 2003; 14: 1240-1254Crossref PubMed Scopus (293) Google Scholar, 5Farge E. Biophys. J. 1995; 69: 2501-2506Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 6Farge E. Ojcius D.M. Subtil A. Dautry-Varsat A. Am. J. Physiol. 1999; 276: C725-C733Crossref PubMed Google Scholar). The fact that yeast expresses five P-type ATPase family members in different compartments of the exocytic and endocytic transport pathways suggests the possibility that all membrane budding from sphingolipid- and cholesterol-rich membranes depends on the mass translocation of membrane phospholipids.The bulk membrane phospholipid in mammalian cells is PC, which constitutes 25-50 mol % of the membrane lipids. However, it is unclear if cells possess mechanisms for the plasma membrane translocation of PC as for PS and PE. PC is a cylindrical lipid with a low tendency to flip across membranes, with a half-time of days in model membranes. Indeed, using natural PC (7van Meer G. Op den Kamp J.A. J. Cell. Biochem. 1982; 19: 193-204Crossref PubMed Scopus (51) Google Scholar, 8Middelkoop E. Lubin B.H. Op den Kamp J.A. Roelofsen B. Biochim. Biophys. Acta. 1986; 855: 421-424Crossref PubMed Scopus (71) Google Scholar, 9Tilley L. Cribier S. Roelofsen B. Op den Kamp J.A. van Deenen L.L. FEBS Lett. 1986; 194: 21-27Crossref PubMed Scopus (126) Google Scholar) or spin-labeled and fluorescent (C6-NBD-) short chain PCs (10Colleau M. Herve P. Fellmann P. Devaux P.F. Chem. Phys. Lipids. 1991; 57: 29-37Crossref PubMed Scopus (94) Google Scholar, 11Zachowski A. Biochem. J. 1993; 294: 1-14Crossref PubMed Scopus (693) Google Scholar), typical half-times for inward translocation have been reported of hours (as compared with minutes for PS) in the erythrocyte membrane as a model plasma membrane. Still, in some cells the short chain PCs displayed rapid inward translocation (12Sleight R.G. Abanto M.N. J. Cell Sci. 1989; 93: 363-374PubMed Google Scholar) in an ATP-dependent and N-ethylmaleimide-sensitive manner (13Pomorski T. Herrmann A. Muller P. van Meer G. Burger K. Biochemistry. 1999; 38: 142-150Crossref PubMed Scopus (34) Google Scholar), as they do in yeast (4Pomorski T. Lombardi R. Riezman H. Devaux P.F. van Meer G. Holthuis J.C. Mol. Biol. Cell. 2003; 14: 1240-1254Crossref PubMed Scopus (293) Google Scholar, 14Grant A.M. Hanson P.K. Malone L. Nichols J.W. Traffic. 2001; 2: 37-50Crossref PubMed Scopus (65) Google Scholar). Either some cells possess a specific inward PC translocator or some aminophospholipid translocases are not strictly specific for PS and PE. In erythrocytes, aminophospholipids also translocate in the opposite, outward direction but much slower than inward: short chain PS and PE displayed ATP-dependent inward and outward transport with half-times of 3 and 35 min, respectively, for the inward direction and 58 and 77 min for the outward direction (15Connor J. Pak C.H. Zwaal R.F. Schroit A.J. J. Biol. Chem. 1992; 267: 19412-19417Abstract Full Text PDF PubMed Google Scholar, 16Bitbol M. Devaux P.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6783-6787Crossref PubMed Scopus (101) Google Scholar). Similarly, the outward movement of C6-NBD-PC was ATP-dependent (15Connor J. Pak C.H. Zwaal R.F. Schroit A.J. J. Biol. Chem. 1992; 267: 19412-19417Abstract Full Text PDF PubMed Google Scholar). First hints for the identity of one PC outward translocator came from the finding that PC secretion into mouse bile depended on the presence of the ATP-binding cassette (ABC) transporter Abcb4 (17Smit J.J. Schinkel A.H. Oude Elferink R.P. Groen A.K. Wagenaar E. van Deemter L. Mol C.A. Ottenhoff R. van der Lugt N.M. van Roon M.A. van der Valk M.A. Offerhaus G.J. Berns A.J. Borst P. Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1318) Google Scholar), and that this liver transporter enhanced transport of newly synthesized PC to the surface of transgenic fibroblasts (18Smith A.J. Timmermans-Hereijgers J.L. Roelofsen B. Wirtz K.W. van Blitterswijk W.J. Smit J.J. Schinkel A.H. Borst P. FEBS Lett. 1994; 354: 263-266Crossref PubMed Scopus (252) Google Scholar). In studies on short chain lipids, human ABCB4 was found to be specific for PC, whereas, unexpectedly, the closely related multidrug transporter ABCB1 translocated a wide variety of short chain lipids (19van Helvoort A. Smith A.J. Sprong H. Fritzsche I. Schinkel A.H. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar, 20Bosch I. Dunussi-Joannopoulos K. Wu R.L. Furlong S.T. Croop J. Biochemistry. 1997; 36: 5685-5694Crossref PubMed Scopus (136) Google Scholar), including the short chain PC platelet activating factor (21Ernest S. Bello-Reuss E. J. Am. Soc. Nephrol. 1999; 10: 2306-2313Crossref PubMed Google Scholar, 22Raggers R.J. Vogels I. van Meer G. Biochem. J. 2001; 357: 859-865Crossref PubMed Scopus (81) Google Scholar). However, in erythrocytes the outward translocation of C6-NBD-PC and -PS was found to be mediated by an alternative ABC transporter, ABCC1 (23Dekkers D.W. Comfurius P. Schroit A.J. Bevers E.M. Zwaal R.F. Biochemistry. 1998; 37: 14833-14837Crossref PubMed Scopus (102) Google Scholar). Because the expression of ABCB4 is rather specific for liver and there is no convincing evidence that ABCB1 and ABCC1 translocate natural, long chain lipids, the question remains whether natural PC is actively translocated across the plasma membrane of non-hepatocytes.Outward translocation of natural PC has been reported in rat and human erythrocytes (24Renooij W. van Golde L.M. Zwaal R.F. van Deenen L.L. Eur. J. Biochem. 1976; 61: 53-58Crossref PubMed Scopus (167) Google Scholar, 25Renooij W. van Golde L.M. Biochim. Biophys. Acta. 1977; 470: 465-474Crossref PubMed Scopus (46) Google Scholar, 26Andrick C. Broring K. Deuticke B. Haest C.W. Biochim. Biophys. Acta. 1991; 1064: 235-241Crossref PubMed Scopus (21) Google Scholar). The process appeared restricted to newly synthesized PC, sensitive to the arginine modifying reagent phenylglyoxal, and insensitive to vanadate (26Andrick C. Broring K. Deuticke B. Haest C.W. Biochim. Biophys. Acta. 1991; 1064: 235-241Crossref PubMed Scopus (21) Google Scholar), suggesting protein-mediated but energy-independent PC translocation as it was also proposed for bile canalicular membranes (27Berr F. Meier P.J. Stieger B. J. Biol. Chem. 1993; 268: 3976-3979Abstract Full Text PDF PubMed Google Scholar). In the present study we re-evaluated the translocation of natural PC from the inside to the outside of the plasma membrane. For this, we optimized two independent assays for measuring the fraction of intracellularly labeled PC that arrived at the cell surface, the hydrolysis of cell surface PC with phospholipase A2 (1Verkleij A.J. Zwaal R.F. Roelofsen B. Comfurius P. Kastelijn D. van Deenen L.L. Biochim. Biophys. Acta. 1973; 323: 178-193Crossref PubMed Scopus (819) Google Scholar) and exchange of outer leaflet PC against liposomal PC by the PC transfer protein (28van Meer G. Poorthuis B.J. Wirtz K.W. Op den Kamp J.A. van Deenen L.L. Eur. J. Biochem. 1980; 103: 283-288Crossref PubMed Scopus (73) Google Scholar). We investigated PC cell surface translocation in erythrocytes and in fibroblasts and present evidence that PC is actively translocated across mammalian plasma membranes with characteristics that would be in accordance with an involvement of ABC transporters.EXPERIMENTAL PROCEDURESMaterials—The radioactive fatty acids [1-14C]arachidonic acid (50 Ci/mol), ([1-14C]- and [U-14C]palmitic acid (50 Ci/mol and 500 Ci/mol, respectively), and [1-14C]oleic acid (50 Ci/mol) were from Amersham Biosciences; l-[palmitoyl-1-14C]carnitine chloride (50 Ci/mol) was from PerkinElmer Life Sciences, [32P]H3PO4 in H2O was from ICN (Zoetermeer, The Netherlands), NBD-labeled lipids were purchased from Avanti Polar Lipids (Alabaster, AL). Chemicals and enzymes, if not indicated otherwise, were from Sigma and used in the highest purity available. Indomethacin was from ICN (Aurora, OH). PSC833 was a kind gift from Novartis Pharma AG (Basel, Switzerland). Ko143 was a kind gift from Alfred Schinkel (NKI, Amsterdam, The Netherlands). Silica TLC plates were from Merck (Darmstadt, Germany), organic solvents were from Riedel de Haën (Darmstadt, Germany), and cell culture media were from Invitrogen.Lipid Analysis—Lipids from intact cells were extracted according to Bligh and Dyer (29Bligh E.G. Dyer W.J. Can. J. Biochem. Phys. 1959; 37: 911-917Crossref PubMed Scopus (42174) Google Scholar), dried under nitrogen, and separated by two-dimensional thin layer chromatography with the first dimension in chloroform, methanol, 25% ammonia (65:25:4), followed by chloroform: methanol:acetone:acetic acid:water (50:20:10:10:5) (30van der Bijl P. Strous G.J. Lopes-Cardozo M. Thomas-Oates J. van Meer G. Biochem. J. 1996; 317: 589-597Crossref PubMed Scopus (51) Google Scholar) (Fig. 1). [32P]Phosphate-labeled lipids were separated before TLC on Accell Plus CM anion exchange SepPak columns (Waters, Etten-Leur, The Netherlands). Briefly, columns were equilibrated with CHCl3:MeOH (2:1), the lipids were loaded in a few drops of CHCl3:MeOH (2:1), and the uncharged lipids were eluted with 4 ml of 1 mm ammonium acetate in CHCl3:MeOH:H2O (3:6:1). 1.2 ml of 4 mm HCl was added and a phase separation was performed. Anionic lipids were eluted with increasing ammonium acetate concentrations. TLC plates were exposed to a phosphorimager screen (BAS-MS or BAS-TR, Fuji Medical Systems, Stanford, CT) for 2 days and scanned with a Personal Molecular Imager FX System (Bio-Rad). The fluorescence of C6-NBD-PC-labeled cells was measured with a STORM imaging system (Amersham Biosciences). Quantifications were performed with Quantity One Software (Bio-Rad). Total phospholipids were quantified by phosphate determination after scraping the iodine-stained lipid spots from two-dimensional TLC plates (31Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2859) Google Scholar).Cells and Animals—Wt1.2 mouse fibroblasts (32Allen J.D. Brinkhuis R.F. van Deemter L. Wijnholds J. Schinkel A.H. Cancer Res. 2000; 60: 5761-5766PubMed Google Scholar) were cultured in Dulbecco's modified Eagle's medium containing Glutamax-I, 4.5 g/liter glucose, and 10% heat-inactivated fetal calf serum under 5% CO2. Human erythrocytes were obtained from healthy volunteers by venipuncture, from anesthetized mice by heart puncture or from the eye background with sodium heparin as anticoagulant. Animal experiments were performed according to the guidelines of the Dutch government concerning animal care. Erythrocytes were collected by centrifugation and the buffy coat was removed by five washes with buffer A (140 mm NaCl, 5 mm KCl, 10 mm Hepes, pH 7.4). Cells were resuspended in buffer A, 15 mm glucose, and used within 2 days. Mouse blood was investigated on the day of blood withdrawal. Blood from the double Abcb1a/1b-/- and triple Abcb1a/1b-/-, Abcc1-/- (33Schinkel A.H. Mayer U. Wagenaar E. Mol C.A. van Deemter L. Smit J.J. van der Valk M.A. Voordouw A.C. Spits H. van Tellingen O. Zijlmans J.M. Fibbe W.E. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4028-4033Crossref PubMed Scopus (861) Google Scholar, 34Wijnholds J. de Lange E.C. Scheffer G.L. van den Berg D.J. Mol C.A. van der Valk M. Schinkel A.H. Scheper R.J. Breimer D.D. Borst P. J. Clin. Investig. 2000; 105: 279-285Crossref PubMed Scopus (340) Google Scholar), Abcb4-/- (17Smit J.J. Schinkel A.H. Oude Elferink R.P. Groen A.K. Wagenaar E. van Deemter L. Mol C.A. Ottenhoff R. van der Lugt N.M. van Roon M.A. van der Valk M.A. Offerhaus G.J. Berns A.J. Borst P. Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1318) Google Scholar), Abcc7-/- (35Ratcliff R. Evans M.J. Cuthbert A.W. MacVinish L.J. Foster D. Anderson J.R. Colledge W.H. Nat. Genet. 1993; 4: 35-41Crossref PubMed Scopus (213) Google Scholar), and Abca1-/- (36Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (461) Google Scholar) knockout mice was generously provided by Alfred Schinkel (NKI, Amsterdam), Ronald Oude Elferink (AMC, Amsterdam), Hugo de Jonge (EUR, Rotterdam), and Folkert Kuipers (RUG, Groningen), respectively. In each experiment, performed in duplicate, three knockout animals were compared with three matched control animals.Energy-depletion of Human Erythrocytes—Human erythrocytes were incubated for 2 h in buffer A containing 50 mm deoxyglucose and 5 mm KF (37Plagemann P.G. Wohlhueter R.M. Kraupp M. Biochim. Biophys. Acta. 1985; 817: 51-60Crossref PubMed Scopus (22) Google Scholar) before a 2-h incubation in the presence of the label, [14C]palmitoylcarnitine (38Arduini A. Mancinelli G. Radatti G.L. Dottori S. Molajoni F. Ramsay R.R. J. Biol. Chem. 1992; 267: 12673-12681Abstract Full Text PDF PubMed Google Scholar) or [14C]arachidonic acid, where we used 0.2 μCi in energy-depleted and 0.02 μCi of [14C]arachidonic acid per 100 nmol of total phospholipid in fresh erythrocytes.Preparation of Resealed Erythrocyte Ghosts—Ghosts were prepared from fresh erythrocytes by hypotonic shock. One volume of the erythrocyte pellet was diluted into 4 volumes of ice-cold lysis buffer (9 mm KCl, 4.5 mm NaCl, 2 mm MgCl2, 0.22 mm EGTA, 1 mm Hepes, pH 7). After 15 min on ice 2 mm ATP or AMP-PNP were added and tonicity was restored by addition of 900 mm KCl, 450 mm NaCl, 20 mm MgCl2, 2 mm EGTA, 100 mm Hepes, pH 7, and cells were resealed for 1 h at 37 °C. Resealed ghosts were collected by centrifugation (10 min, 3,200 × g) and washed three times with buffer A.Metabolic Labeling—Fibroblasts in 6-well plates were preincubated for 20 min with 1 μg/ml brefeldin A to block vesicular traffic, with or without candidate translocation inhibitors. The cells were labeled for the indicated times with 20 μCi/well [32P]phosphate (about 9 × 105 cells), washed with PBS, and chased for different time periods before analysis with a cell surface PC assay. Erythrocytes were preincubated for 20 min with or without inhibitors, labeled with trace amounts of [14C]arachidonic, -oleic, or -palmitic acid (0.1-1 nmol/100 nmol of erythrocyte lipids) or with [14C]palmitoylcarnitine (1.5 nmol/100 nmol of erythrocyte lipids), washed with 2% BSA and twice with buffer A, and chased for the indicated time periods with or without inhibitors before analysis with a cell surface PC assay.PLA2Cell Surface Assay—Cells (150 nmol of phospholipid) were incubated in 500 μl of buffer A plus 10 mm CaCl2 and 50 IU bee venom phospholipase A2 (PLA2) for 5 min at 37 °C. Hemolysis as the absorption of the cell supernatant at 540 nm in a spectrophotometer was below 4%. Lipids were quantified after two-dimensional TLC analysis. [14C]PC exposure at the erythrocyte surface was expressed as % decrease of [14C]PC in the PLA2-treated sample as compared with the identically treated control but without PLA2. If [14C]PC in the PLA2-treated sample was not decreased but slightly higher than in the control, this way of quantification generates negative values. Alternatively, [14C]PC exposure at the erythrocyte surface was expressed as % of 1-[14C]palmitoyl lyso-PC of the total 1-[14C]palmitoyl-PC in the control without PLA2. For the quantification of total 1-[14C]palmitoyl-PC in the control, lipids were extracted, dried under nitrogen, and dissolved in 10 μl of diethyl ether, added to 1 ml of buffer A plus 10 mm CaCl2, containing 1% BSA and 20 IU PLA2, and incubated for 30 min at 37 °C, after which 1-[14C]palmitoyl lyso-PC was quantified. In fibroblasts, [32P]PC at the cell surface was measured as % of [32P]lyso-PC in the PLA2-treated sample from total [32P]PC ([32P]lyso-PC + [32P]PC) in the control without PLA2.PC Transfer Protein (PCTP) Cell Surface Assay—PCTP (a kind gift from K. Wirtz (CBLE, Utrecht)) (39Kamp H.H. Wirtz K.W. Methods Enzymol. 1974; 32: 140-146Crossref PubMed Scopus (71) Google Scholar), was stored in 50% glycerol, which was removed before the assay by using Centricon YM 10 filter units (Millipore, Etten-Leur, The Netherlands). For investigating the outward translocation of [14C]PC, small unilamellar vesicles (SUVs) were generated by sonication on ice (18Smith A.J. Timmermans-Hereijgers J.L. Roelofsen B. Wirtz K.W. van Blitterswijk W.J. Smit J.J. Schinkel A.H. Borst P. FEBS Lett. 1994; 354: 263-266Crossref PubMed Scopus (252) Google Scholar) of egg PC:cholesterol:egg PS (50: 50:1, mol/mol). The amount of SUVs in the cell pellet was below 2% as measured by [14C]cholesterol ester as a non-exchangeable SUV marker. Cells containing 100 nmol of phospholipid were labeled for 40 min with [14C]arachidonate, washed with buffer A, 2% BSA, and twice with buffer A and incubated at 37 °C in 500 μl of buffer A, 15 mm glucose, 3 nmol of PCTP, a 10-fold excess of SUV PC, and a 20-fold excess of cold arachidonate under slow rotation. After the incubation, the cells were washed three times in buffer A containing 1% glycerol. The supernatants were pooled and the lipids of cells and supernatants were analyzed. Cell surface [14C]PC was quantified as [14C]PC in the PCTP supernatant as % of total [14C]PC in supernatant plus cells. In [14C]PC inward translocation studies, SUVs were generated by sonication of (a) PC isolated from [14C]arachidonate-labeled erythrocytes:cholesterol: egg PS (50:50:1) or (b) [32P]PC isolated from mouse fibroblasts:cholesterol:egg PS (50:50:1). Erythrocytes containing 100 nmol of phospholipid were incubated at 37 or 4 °C in 500 μl of buffer A, 15 mm glucose, 3 nmol of PCTP and SUVs. The ratio of total erythrocyte PC to SUV PC was between 1:10 and 1:2. After the incubation, the erythrocytes were washed 3 times with buffer A, 1% glycerol before further incubation and a PLA2 assay.Outward Translocation of C6-NBD-PC—C6-NBD-PC cell surface translocation was measured essentially as described by Connor et al. (15Connor J. Pak C.H. Zwaal R.F. Schroit A.J. J. Biol. Chem. 1992; 267: 19412-19417Abstract Full Text PDF PubMed Google Scholar). 1 ml of packed human erythrocytes were incubated for 90 min at 37 °C with 15-20 nmol of C6-NBD-PC in buffer A, 15 mm glucose, containing 1 m ethanol to reversibly accelerate phospholipid flip (40Schwichtenhovel C. Deuticke B. Haest C.W. Biochim. Biophys. Acta. 1992; 1111: 35-44Crossref PubMed Scopus (45) Google Scholar). Because all samples were identically labeled with C6-NBD-PC in the presence of 1 m ethanol, differences in translocation rates are irrespective of the ethanol. C6-NBD-PC still at the cell surface was removed by two washes with 20 volumes of buffer A, 2% BSA, for 2 min at room temperature. Outward translocation of the lipid analog was measured by incubating the cells at 37 °C in buffer A, 15 mm glucose, with or without candidate inhibitors for various times. Cell surface C6-NBD-PC was depleted with BSA as described above. Lipids in the cells and BSA washes were extracted and separated by one-dimensional TLC in acidic solvent. C6-NBD-PC cell surface exposure was measured as % of C6-NBD-PC in the supernatant from total C6-NBD-PC in cells plus supernatant. % C6-NBD-FA was below 10% of total NBD fluorescence and increased during the chase by less than 5%, independent of the inhibitors.RESULTSTo investigate the outward translocation of natural PC across the plasma membrane of mammalian cells, we first optimized two independent PC cell surface assays in erythrocytes. Here, the absence of intracellular membranes facilitates the analysis of transport processes at the plasma membrane and allows to optimally control cell surface assay conditions. If the assay induced disturbances at the plasma membrane, these would be detectable by changes in total lipid asymmetry, erythrocyte shape, and by increased hemolysis. The high percentage of PC at the erythrocyte surface is favorable for its quantification.Assays for the Translocation of Natural PCGeneration of Labeled PC at the Inside of the Plasma Membrane—The investigation of the outward translocation of natural PC depends on placing a labeled PC at the inside of the cell and an assay for its appearance on the outside. Mature erythrocytes are not able to synthesize lipids de novo but exogenous fatty acids are rapidly taken up and coupled to pre-existing lyso-PC via an ATP-dependent acyl-CoA synthetase and an acyl-CoA:lysophospholipid acyltransferase at the inside of the plasma membrane (24Renooij W. van Golde L.M. Zwaal R.F. van Deenen L.L. Eur. J. Biochem. 1976; 61: 53-58Crossref PubMed Scopus (167) Google Scholar, 25Renooij W. van Golde L.M. Biochim. Biophys. Acta. 1977; 470: 465-474Crossref PubMed Scopus (46) Google Scholar, 26Andrick C. Broring K. Deuticke B. Haest C.W. Biochim. Biophys. Acta. 1991; 1064: 235-241Crossref PubMed Scopus (21) Google Scholar). The sidedness of PC labeling was demonstrated experimentally. Insertion of 5 mol % of lyso-PC into the outer leaflet of the erythrocyte membrane enhanced the incorporation of [14C]arachidonic acid into PC, but not into PE or PS. This effect was only seen after increasing chase periods and was after 5 min of chase 111%, after 15 min 140%, and after 25 min 203% of PC labeling in the control without exogenous lyso-PC. In contrast, when lyso-PC was offered to both sides of the plasma membrane during hypo-osmotic shock, PC labeling increased to 2300% after 15 min, supporting bulk PC labeling at the inside. The stimulatory effect of exogenous lyso-PC on PC labeling in intact erythrocytes is best explained by slow inward movement of lyso-PC.Detecting Labeled PC at the Cell Surface—Two methods have been used to measure the transbilayer distribution of PC across the erythrocyte membrane, hydrolysis of surface PC by PLA2 (1Verkleij A.J. Zwaal R.F. Roelofsen B. Comfurius P. Kastelijn D. van Deenen L.L. Biochim. Biophys. Acta. 1973; 323: 178-193Crossref PubMed Scopus (819) Google Scholar, 24Renooij W. van Golde L.M. Zwaal R.F. van Deenen L.L. Eur. J. Biochem. 1976; 61: 53-58Crossref PubMed Scopus (167) Google Scholar, 25Renooij W. van Golde L.M. Biochim. Biophys. Acta. 1977; 470: 465-474Crossref PubMed Scopus (46) Google Scholar, 26Andrick C. Broring K. Deuticke B. Haest C.W. Biochim. Biophys. Acta. 1991; 1064: 235-241Crossref PubMed Scopus (21) Google Scholar) and exchange of outer leaflet PC against liposomal PC by a lipid transfer protein (28van Meer G. Poorthuis B.J. Wirtz K.W. Op den Kamp J.A. van Deenen L.L. Eur. J. Biochem. 1980; 103: 283-288Crossref PubMed Scopus (73) Google Scholar, 41Crain R.C. Zilversmit D.B. Biochim. Biophys. Acta. 1980; 620: 37-48Crossref PubMed Scopus (40) Google Scholar).First, we optimized the PLA2 assay to reduce incubation times and allow the immediate detection of newly synthesized PC at the cell surface. Incubation of a 50-μl erythrocyte pellet with 50 IU PLA2 in 500 μl of buffer A, 10 mm CaCl2 for 5 min at 37 °C resulted in almost complete degradation of cell surface PC, namely 92 ± 1.4% of [32P]PC that had been introduced into the outer bilayer leaflet by PCTP at 4 °C (Table I). The generated degradation pattern of total phospholipid was in agreement with the literature on erythrocyte lipid asymmetry. No breakdown of PS was detected, demonstrating restriction of PLA2 activity to the surface lipids, unless 1% BSA was present with PLA2, causing hemolysis by extracting free fatty acids and lysolipids. The PLA2 hydrolysis pattern of the [14C]fatty acid-labeled phosphoglycerolipids (Fig. 1) largely reflected that of total lipids, showing full equilibration of the labeled lipids after overnight chase. When analyzing the cell surface exposure of [14C]arachidonoyl-PC and [14C]oleoyl-PC after 30 min labeling, we already found 37 ± 4 and 28 ± 17% at the cell surface, which increased to 56 ± 7 and 53 ± 6% after an overnight chase, respectively, suggesting fast outward translocation of PC with a half-time below 30 min. Also [14C]PE rapidly appeared at the cell surface, however, the large standard deviations inherent to the determination of the minor fractions of" @default.
• W2125973635 created "2016-06-24" @default.
• W2125973635 creator A5014241448 @default.
• W2125973635 creator A5027488443 @default.
• W2125973635 creator A5030256996 @default.
• W2125973635 creator A5066790177 @default.
• W2125973635 date "2004-08-01" @default.
• W2125973635 modified "2023-10-15" @default.
• W2125973635 title "Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells" @default.
• W2125973635 cites W1484056972 @default.
• W2125973635 cites W1512735145 @default.
• W2125973635 cites W1580615597 @default.
• W2125973635 cites W1717245686 @default.
• W2125973635 cites W1762647466 @default.
• W2125973635 cites W1813431637 @default.
• W2125973635 cites W1837904833 @default.
• W2125973635 cites W1941920247 @default.
• W2125973635 cites W1963552073 @default.
• W2125973635 cites W1966330055 @default.
• W2125973635 cites W1968968981 @default.
• W2125973635 cites W1970458460 @default.
• W2125973635 cites W1970830865 @default.
• W2125973635 cites W1970951117 @default.
• W2125973635 cites W1974739502 @default.
• W2125973635 cites W1977986845 @default.
• W2125973635 cites W1979233174 @default.
• W2125973635 cites W1980224025 @default.
• W2125973635 cites W1984376924 @default.
• W2125973635 cites W1991504728 @default.
• W2125973635 cites W1992489111 @default.
• W2125973635 cites W1992928034 @default.
• W2125973635 cites W1995532798 @default.
• W2125973635 cites W1996477510 @default.
• W2125973635 cites W1996496621 @default.
• W2125973635 cites W2000266304 @default.
• W2125973635 cites W2003709444 @default.
• W2125973635 cites W2005859080 @default.
• W2125973635 cites W2019780382 @default.
• W2125973635 cites W2028744351 @default.
• W2125973635 cites W2028899000 @default.
• W2125973635 cites W2030747544 @default.
• W2125973635 cites W2038791554 @default.
• W2125973635 cites W2043731138 @default.
• W2125973635 cites W2044531031 @default.
• W2125973635 cites W2044656058 @default.
• W2125973635 cites W2046338633 @default.
• W2125973635 cites W2047330360 @default.
• W2125973635 cites W2050410538 @default.
• W2125973635 cites W2053176488 @default.
• W2125973635 cites W2054680310 @default.
• W2125973635 cites W2059428912 @default.
• W2125973635 cites W2060759428 @default.
• W2125973635 cites W2064790787 @default.
• W2125973635 cites W2067541521 @default.
• W2125973635 cites W2068813676 @default.
• W2125973635 cites W2076236539 @default.
• W2125973635 cites W2076988559 @default.
• W2125973635 cites W2080075338 @default.
• W2125973635 cites W2085715843 @default.
• W2125973635 cites W2087530953 @default.
• W2125973635 cites W2087774895 @default.
• W2125973635 cites W2092736567 @default.
• W2125973635 cites W2094365838 @default.
• W2125973635 cites W2107290681 @default.
• W2125973635 cites W2110696115 @default.
• W2125973635 cites W2116955854 @default.
• W2125973635 cites W2117904740 @default.
• W2125973635 cites W2118667947 @default.
• W2125973635 cites W2119468178 @default.
• W2125973635 cites W2135585405 @default.
• W2125973635 cites W2153338237 @default.
• W2125973635 cites W2164477711 @default.
• W2125973635 cites W2166153381 @default.
• W2125973635 cites W2168379147 @default.
• W2125973635 cites W2169542233 @default.
• W2125973635 cites W2172134989 @default.
• W2125973635 cites W2283648576 @default.
• W2125973635 cites W2300552860 @default.
• W2125973635 cites W4236512430 @default.
• W2125973635 cites W4237311797 @default.
• W2125973635 doi "https://doi.org/10.1074/jbc.m401751200" @default.
• W2125973635 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15175345" @default.
• W2125973635 hasPublicationYear "2004" @default.
• W2125973635 type Work @default.
• W2125973635 sameAs 2125973635 @default.
• W2125973635 citedByCount "32" @default.
• W2125973635 countsByYear W21259736352013 @default.
• W2125973635 countsByYear W21259736352014 @default.
• W2125973635 countsByYear W21259736352015 @default.
• W2125973635 countsByYear W21259736352016 @default.
• W2125973635 countsByYear W21259736352017 @default.
• W2125973635 countsByYear W21259736352018 @default.
• W2125973635 countsByYear W21259736352022 @default.
• W2125973635 countsByYear W21259736352023 @default.
• W2125973635 crossrefType "journal-article" @default.
• W2125973635 hasAuthorship W2125973635A5014241448 @default.
• W2125973635 hasAuthorship W2125973635A5027488443 @default.
• W2125973635 hasAuthorship W2125973635A5030256996 @default.

• next