Activation of Membrane Cholesterol by Displacement from Phospholipids

We tested the hypothesis that certain membrane-intercalating agents increase the chemical activity of cholesterol by displacing it from its low activity association with phospholipids. Octanol, 1,2-dioctanoyl-sn-glycerol (a diglyceride), and N-hexanoyl-d-erythrosphingosine (a ceramide) were shown to increase both the rate of transfer and the extent of equilibrium partition of human red blood cell cholesterol to methyl-β-cyclodextrin. These agents also promoted the interaction of the sterol with two cholesterol-specific probes, cholesterol oxidase and saponin. Expanding the pool of bilayer phospholipids with lysophosphatides countered these effects. The three intercalators also protected the red cells against lysis by cholesterol depletion as if substituting for the extracted sterol. As is the case for excess plasma membrane cholesterol, treating human fibroblasts with octanol, diglyceride, or ceramide stimulated the rapid inactivation of their hydroxymethylglutaryl-CoA reductase, presumably through an increase in the pool of endoplasmic reticulum cholesterol. These data supported the stated hypothesis and point to competition between cholesterol and endogenous and exogenous intercalators for association with membrane phospholipids. We also describe simple screens using red cells in a microtiter well format to identify intercalating agents that increase or decrease the activity of membrane cholesterol. We tested the hypothesis that certain membrane-intercalating agents increase the chemical activity of cholesterol by displacing it from its low activity association with phospholipids. Octanol, 1,2-dioctanoyl-sn-glycerol (a diglyceride), and N-hexanoyl-d-erythrosphingosine (a ceramide) were shown to increase both the rate of transfer and the extent of equilibrium partition of human red blood cell cholesterol to methyl-β-cyclodextrin. These agents also promoted the interaction of the sterol with two cholesterol-specific probes, cholesterol oxidase and saponin. Expanding the pool of bilayer phospholipids with lysophosphatides countered these effects. The three intercalators also protected the red cells against lysis by cholesterol depletion as if substituting for the extracted sterol. As is the case for excess plasma membrane cholesterol, treating human fibroblasts with octanol, diglyceride, or ceramide stimulated the rapid inactivation of their hydroxymethylglutaryl-CoA reductase, presumably through an increase in the pool of endoplasmic reticulum cholesterol. These data supported the stated hypothesis and point to competition between cholesterol and endogenous and exogenous intercalators for association with membrane phospholipids. We also describe simple screens using red cells in a microtiter well format to identify intercalating agents that increase or decrease the activity of membrane cholesterol. Sterols associate in roughly equimolar proportions with the phospholipids in artificial membranes (1Huang J. Buboltz J.T. Feigenson G.W. Biochim. Biophys. Acta. 1999; 1417: 89-100Crossref PubMed Scopus (370) Google Scholar, 2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar). The sterols associated with the phospholipids are held at a low chemical activity. In sharp contrast, sterol molecules in excess of the capacity of the phospholipids are free and have a much higher chemical activity (3Huang J. Feigenson G.W. Biophys. J. 1999; 76: 2142-2157Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 4McConnell H.M. Radhakrishnan A. Biochim. Biophys. Acta. 2003; 1610: 159-173Crossref PubMed Scopus (351) Google Scholar). The high fugacity (escape potential) of the superthreshold sterol promotes its exit from the membrane, thereby tending to restore the balance between the sterols and phospholipids (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar). Most of the cholesterol in mammalian cells is in the plasma membranes, where its abundance appears to be maintained homeostatically near molar equivalence with the phospholipids (5Lange Y. Swaisgood M.H. Ramos B.V. Steck T.L. J. Biol. Chem. 1989; 264: 3786-3793Abstract Full Text PDF PubMed Google Scholar, 6Lange Y. Ye J. Rigney M. Steck T. J. Biol. Chem. 2000; 275: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Furthermore, cholesterol in excess of the physiological set point appears to have a high chemical activity. One indicator of this activity is the sharp increase in the susceptibility to cholesterol oxidase of human red blood cell and fibroblast plasma membranes that have been augmented slightly with cholesterol (7Lange Y. Cutler H.B. Steck T.L. J. Biol. Chem. 1980; 255: 9331-9337Abstract Full Text PDF PubMed Google Scholar, 8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). We interpret this effect to signify that, above the threshold, cholesterol molecules are free of constraint by phospholipids and more frequently bob out of the plane of the bilayer, where the peripherally bound enzyme can access them (see Ref. 9Chen X. Wolfgang D.E. Sampson N.S. Biochemistry. 2000; 39: 13383-13389Crossref PubMed Scopus (27) Google Scholar). Similarly, the rate of transfer to cyclodextrin 2The abbreviations and trivial names used are:cyclodextrinmethyl-β-cyclodextrinceramideN-hexanoyl-d-erythro-sphingosinediglyceride1,2-dioctanoyl-sn-glycerol25HC25-hydroxycholesterolPBS150 mM NaCl, 5 mM NaPi (pH 7.5)HMGhydroxymethylglutarylHPLChigh pressure liquid chromatography 2The abbreviations and trivial names used are:cyclodextrinmethyl-β-cyclodextrinceramideN-hexanoyl-d-erythro-sphingosinediglyceride1,2-dioctanoyl-sn-glycerol25HC25-hydroxycholesterolPBS150 mM NaCl, 5 mM NaPi (pH 7.5)HMGhydroxymethylglutarylHPLChigh pressure liquid chromatography of membrane cholesterol in slight excess of normal is dramatically increased (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar, 8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). This effect could reflect a high frequency of transient projections of membrane sterol molecules into the aqueous phase where they can be captured by the acceptor (10Steck T.L. Ye J. Lange Y. Biophys. J. 2002; 83: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Finally, augmenting the plasma membranes of fibroblasts with a slight excess of cholesterol drives some to the endoplasmic reticulum, perhaps because of its high escape potential. Multiple regulatory elements in the endoplasmic reticulum sense the elevated cholesterol and respond by reducing the cell cholesterol level (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar, 20Lange Y. Ye J. Rigney M. Steck T.L. J. Lipid Res. 1999; 40: 2264-2270Abstract Full Text Full Text PDF PubMed Google Scholar). methyl-β-cyclodextrin N-hexanoyl-d-erythro-sphingosine 1,2-dioctanoyl-sn-glycerol 25-hydroxycholesterol 150 mM NaCl, 5 mM NaPi (pH 7.5) hydroxymethylglutaryl high pressure liquid chromatography methyl-β-cyclodextrin N-hexanoyl-d-erythro-sphingosine 1,2-dioctanoyl-sn-glycerol 25-hydroxycholesterol 150 mM NaCl, 5 mM NaPi (pH 7.5) hydroxymethylglutaryl high pressure liquid chromatography These data suggest that a simple physicochemical mechanism plays a central role in the homeostasis of eukaryotic cell cholesterol: the steady-state level of the sterol in the plasma membrane is set by the capacity of its phospholipids (perhaps along with other relevant polar lipids). Passive and active adjustments made in response to the chemical potential of the cholesterol in excess of the equivalence point serve to restore the resting level of plasma membrane cholesterol (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar, 8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). There is evidence that the association of cholesterol with phospholipids can be perturbed so as to increase its chemical activity. In particular, it has been suggested that ceramides and diglycerides, physiological membrane-intercalating amphipathic alcohols, displace cholesterol from bilayer phospholipids (11Zitzer A. Bittman R. Verbicky C.A. Erukulla R.K. Bhakdi S. Weis S. Valeva A. Palmer M. J. Biol. Chem. 2001; 276: 14628-14633Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). In addition, octanol not only stimulates the oxidation of plasma membrane cholesterol by cholesterol oxidase and its transfer to cyclodextrin but also sends the sterol to the endoplasmic reticulum (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar, 13Lange Y. Matthies H. Steck T.L. Biochim. Biophys. Acta. 1984; 769: 551-562Crossref PubMed Scopus (55) Google Scholar). We therefore sought to test the emergent hypothesis that these intercalating alcohols increase the chemical activity of cholesterol by displacing it from phospholipids. Materials—1,2-Dioctanoyl-sn-glycerol and N-hexanoyl-d-erythrosphingosine were obtained from Avanti. l-α-lyso(palmitoyl)phosphatidyl-l-serine, egg yolk lysophosphatidylcholine, 25HC, cyclodextrin, octanol, and cholesterol oxidase (Streptomyces sp) were all from Sigma. We purchased 1α,2α-[3H]cholesterol from Amersham Biosciences. Blood was donated by a healthy human volunteer. Red blood cells were separated from the plasma and buffy coat and washed thrice with 10 volumes of PBS. Chemical Assay of Red Cell Susceptibility to Cholesterol Oxidase—As described in the figure legends, red cells in PBS were preincubated in microcentrifuge tubes for 5 min at 37 °C with agents or a solvent control (1% v/v ethanol). The samples were then incubated at 37 °C for 1–2 h with cholesterol oxidase, chilled on ice, and centrifuged for 5 min to pellet the cells and any membranes released by lysis during the incubation. Supernatants were retained for the assay of hemolysis, and ghosts were prepared from the pellets (14Steck T.L. Kant J.A. Methods Enzymol. 1974; 31: 172-180Crossref PubMed Scopus (893) Google Scholar). Ghost cholesterol was assayed fluorimetrically with Amplex Red (Molecular Probes); cholesterol oxidation was calculated from the difference between enzyme-treated and control cells. These results agreed well with analysis by HPLC (15Lange Y. Echevarria F. Steck T.L. J. Biol. Chem. 1991; 266: 21439-21443Abstract Full Text PDF PubMed Google Scholar). Hemolysis Assay of Red Cell Susceptibility to Cholesterol Oxidase— Red cells in 96-well plates were preincubated with the agents for 5 min at 37 °C. Cholesterol oxidase was added for a 1–2-h incubation at 37 °C. The samples were mixed by inversion, and their light scattering was in a as the at the cells were to or the plates centrifuged and then fibroblasts were maintained in and were incubated in to the of hydroxymethylglutaryl-CoA (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). transfer of cholesterol from red cells to cyclodextrin was as described (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar, T.L. Ye J. Lange Y. Biophys. J. 2002; 83: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). the of red cell cholesterol, cells were incubated with cyclodextrin or to decrease or increase their cholesterol, as described (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). Cholesterol was by HPLC (15Lange Y. Echevarria F. Steck T.L. J. Biol. Chem. 1991; 266: 21439-21443Abstract Full Text PDF PubMed Google or with Amplex was with using as of Red Cell Cholesterol by to have shown that the rate of of red cell membrane cholesterol to excess cyclodextrin is stimulated both by the cholesterol to above the level and by with octanol (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). As shown in ceramide and also this transfer The rate for transfer was increased in both of the The hypothesis test that it is not the rate of of red cell cholesterol to cyclodextrin that be increased by its from phospholipids but the partition We therefore the effect of the intercalators on the equilibrium of cholesterol between red cells and ceramide and were to increase the partition to cyclodextrin by The effect was not This was because only the of cholesterol molecules that were active and not the have a high escape potential. Furthermore, the of cyclodextrin used was to cell Finally, cholesterol partition have been if the intercalators bound to the cyclodextrin as well as the octanol to cyclodextrin could it not the equilibrium partition of cholesterol not octanol cholesterol transfer in (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). of Red Cell Cholesterol with Cholesterol cholesterol in red cells is to cholesterol its susceptibility is bilayer cholesterol is increased slightly and also the cells are with octanol (7Lange Y. Cutler H.B. Steck T.L. J. Biol. Chem. 1980; 255: 9331-9337Abstract Full Text PDF PubMed Google Scholar, 13Lange Y. Matthies H. Steck T.L. Biochim. Biophys. Acta. 1984; 769: 551-562Crossref PubMed Scopus (55) Google Scholar). that treating red cells with three other alcohols, diglyceride, and 25HC, also the oxidation of their cholesterol. at a molar equivalence to the red cell ceramide and were at molar The of the more octanol a excess cholesterol only a was In all the was of the effect of cholesterol (7Lange Y. Cutler H.B. Steck T.L. J. Biol. Chem. 1980; 255: 9331-9337Abstract Full Text PDF PubMed Google Scholar, 8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). The oxidation of their membrane cholesterol red cells to This a to cholesterol that with high the of cholesterol oxidation by the intercalators as that by chemical analysis in has been that the of cholesterol oxidase on the chemical activity of cholesterol in the membrane, in the of the sterol not associated with phospholipids (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). that the membrane the pool of active cholesterol and thereby cholesterol oxidase This was with lysophosphatidylcholine, a membrane-intercalating Y. Matthies H. Steck T.L. Biochim. Biophys. Acta. 1984; 769: 551-562Crossref PubMed Scopus (55) Google Scholar). the mechanism by intercalators stimulated the cholesterol oxidase in and the of cholesterol from augmenting the pool with a slight excess of the activity of cholesterol oxidase promoted by these this of Red Cell Cholesterol with agents on cells in a for the M. S. T. U. J. 1984; PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google Scholar). We therefore tested the lysis of red blood cells by on the activity of excess cholesterol. This not to be the case cells of of their sterol the at active cholesterol could be the of for lysis with membrane cholesterol. This suggested a between the and the sterol. have to with phospholipids for association with the sterol. In that the intercalators lysis by displacing cholesterol from the phospholipids. This is supported by the results shown in and for octanol, and at that of the membrane cholesterol also promoted this effect reflect the of between and of from the in and is that lysophosphatides into the membrane the of the intercalating alcohols to by saponin. We this to be the case with octanol, diglyceride, and ceramide not therefore that not on active cholesterol it a for cholesterol from membrane phospholipids. of for Cholesterol in Red is well that sterols as cholesterol presumably through association with the of the M. Biophys. 1991; PubMed Scopus Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the of sterols from phospholipids by intercalators competition for they for the cholesterol from red cells by cyclodextrin this by that octanol, and all the by the membranes of cholesterol. of the of on has been to increase the level of endoplasmic reticulum cholesterol in fibroblasts (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). was that the mechanism this effect the of plasma membrane cholesterol through from its association with phospholipids (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). test this the effect of other intercalators on the activity of the endoplasmic reticulum This enzyme is in response to in plasma membrane cholesterol activity (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). We that, as all intercalators the activity of during a incubation The effect of is it could reflect the of to increase the level of endoplasmic reticulum cholesterol Y. Ye J. Rigney M. Steck T.L. J. Lipid Res. 1999; 40: 2264-2270Abstract Full Text Full Text PDF PubMed Google Scholar). its in this assay is also to its to the of the enzyme J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the other the other three agents are not to to reductase, and they not its activity added to cell shown but Ref. H. J. Lipid Res. 1991; Full Text PDF PubMed Google Scholar). Furthermore, have that both octanol (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google and J. and T. increase endoplasmic reticulum cholesterol. could not be tested because it the activity the assay Biochim. Biophys. Acta. PubMed Scopus Google Scholar). As a of the of their that the of octanol and on activity were by reducing plasma membrane cholesterol with cyclodextrin to their This presumably not be the case if the intercalators through a of cholesterol. is therefore to that intercalating alcohols that displace cholesterol from the plasma membrane increase its abundance in the endoplasmic reticulum and thereby the for on these it to that a of be of membrane cholesterol by displacing it from bilayer phospholipids. a of could in the of the made between and membrane it also be to identify agents that associate with as by for their to the of cholesterol or of the intercalating alcohols of In red cells were preincubated in microtiter with intercalating and then the of cholesterol oxidase to was As from the results in all three agents stimulated the by cholesterol with the intercalators also promoted red cell lysis by Finally, agents that cholesterol activity can be has been that membrane cholesterol by a cells to oxidation by cholesterol oxidase (7Lange Y. Cutler H.B. Steck T.L. J. Biol. Chem. 1980; 255: 9331-9337Abstract Full Text PDF PubMed Google Scholar). and countered this presumably because they associated with the excess cholesterol and thereby its chemical activity. that the agents protected against in this of This the of the chemical activity of cholesterol on the of the bilayer in human red blood cells. has been suggested that the chemical activity of membrane cholesterol is low by its association with bilayer phospholipids (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar, J. Feigenson G.W. Biophys. J. 1999; 76: 2142-2157Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Cholesterol in excess of the membrane with it can associate be have a high and therefore have a potential to exit the The chemical and of the of and free membrane cholesterol have to be an of increase of sterol activity a that is to be the point of the are an increased rate of cholesterol to cyclodextrin and an increased susceptibility of cholesterol to oxidation by cholesterol oxidase (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar, Y. Cutler H.B. Steck T.L. J. Biol. Chem. 1980; 255: 9331-9337Abstract Full Text PDF PubMed Google Scholar, 8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). indicator of cholesterol activity is the of the cholesterol of fibroblast endoplasmic reticulum in response to in plasma membrane cholesterol Y. Ye J. Rigney M. Steck T.L. J. Lipid Res. 1999; 40: 2264-2270Abstract Full Text Full Text PDF PubMed Google Scholar). also be relevant that the of with high to and to plasma membranes in cholesterol is in slight excess of a near the physiological level Y. M. S. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, Y. M. M. M. S. Y. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). There are two for membrane phospholipids the chemical activity of cholesterol. One that the of bilayer as and sterol molecules from with the aqueous phase (1Huang J. Buboltz J.T. Feigenson G.W. Biochim. Biophys. Acta. 1999; 1417: 89-100Crossref PubMed Scopus (370) Google Scholar, J. Feigenson G.W. Biophys. J. 1999; 76: 2142-2157Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). In this sterol molecules not with the phospholipids but are into by their to Cholesterol molecules in excess of the of be to the phase and therefore have a high chemical activity. In the the excess cholesterol a of (1Huang J. Buboltz J.T. Feigenson G.W. Biochim. Biophys. Acta. 1999; 1417: 89-100Crossref PubMed Scopus (370) Google Scholar). In active cholesterol have free in the bilayer, the evidence that it with in the aqueous phase oxidase and as well as the membrane The hypothesis that membrane sterols and phospholipids associate as (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar, 4McConnell H.M. Radhakrishnan A. Biochim. Biophys. Acta. 2003; 1610: 159-173Crossref PubMed Scopus (351) Google Scholar). to the evidence for this is the that the transfer of cholesterol to is associated with a presumably and a H. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the of phospholipids with for X. London E. Biochemistry. 2000; 39: PubMed Scopus Google Scholar, X. Bittman R. London E. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google the other a plasma membrane with for cholesterol, not in its interaction with cholesterol A. Biophys. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Biophys. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). therefore be that the of the sterols and and are not or the be and as by a E. Biophys. J. 2002; 83: Full Text Full Text PDF PubMed Scopus Google Scholar). and the of liquid that to but not in phase of E. Biol. 2002; PubMed Scopus Google The relevant as in the is that a of phospholipids have a capacity for cholesterol, the chemical activity of the free sterol increase (2Radhakrishnan A. McConnell H.M. Biochemistry. 2000; 39: 8119-8124Crossref PubMed Scopus (140) Google Scholar, 8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). this J. Chem. PubMed Scopus Google Scholar). that agents that free bilayer cholesterol of its association with phospholipids increase its chemical activity. an was from that ceramide and the of cholesterol-specific (11Zitzer A. Bittman R. Verbicky C.A. Erukulla R.K. Bhakdi S. Weis S. Valeva A. Palmer M. J. Biol. Chem. 2001; 276: 14628-14633Abstract Full Text Full Text PDF PubMed Scopus (69) Google and displace cholesterol from in bilayer London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). with the mechanism were that octanol promoted the susceptibility of cholesterol to cholesterol oxidase and the of cholesterol to both octanol and an increase in the cholesterol of the endoplasmic reticulum of fibroblasts (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar, 13Lange Y. Matthies H. Steck T.L. Biochim. Biophys. Acta. 1984; 769: 551-562Crossref PubMed Scopus (55) Google Scholar, 20Lange Y. Ye J. Rigney M. Steck T.L. J. Lipid Res. 1999; 40: 2264-2270Abstract Full Text Full Text PDF PubMed Google Scholar). the emergent the tested membrane-intercalating alcohols diglyceride, and in a the human red cell The were the of cholesterol to cyclodextrin to by and the susceptibility of cholesterol to cholesterol oxidase and In the the effect of a slight excess of cholesterol a membrane-intercalating sterols can be from their association with membrane phospholipids is a We as the that for extracted cholesterol in the cells from lysis and that lysophosphatides the of the intercalators to the of cholesterol oxidase and as they excess cholesterol Y. Matthies H. Steck T.L. Biochim. Biophys. Acta. 1984; 769: 551-562Crossref PubMed Scopus (55) Google Scholar). the lysophosphatides associate with the cholesterol by the intercalators and thereby its chemical its susceptibility to the enzyme (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). is also that the lysophosphatides associate to some with the intercalators to their effect on cholesterol There is also evidence for competition between octanol and cholesterol for association with F. Biochemistry. PubMed Scopus Google Scholar). As a sterol presumably for bilayer cholesterol and because it as well as displace cholesterol, it was not of central in this more was the evidence that as from cholesterol as are diglycerides, and octanol not only displace the sterol from phospholipids but for it in bilayer In with the that these cholesterol not but that they into with the phospholipids through of the and the These data the that of the by the of sterols could be their to in the of by the transient association of endogenous and exogenous intercalators with phospholipids. at a of endogenous and exogenous intercalators be to for sterols physiological and are an London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, A. R. M. J. J. 2003; PubMed Scopus Google Scholar). Finally, results have on the of cholesterol In the the high escape potential of the molecules of plasma membrane cholesterol not associated with the phospholipids of the bilayer sterol to its resting a simple in the case of the red blood the physiological level of cholesterol molar equivalence with the maintained by a equilibrium with plasma (10Steck T.L. Ye J. Lange Y. Biophys. J. 2002; 83: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, J. PubMed Scopus Google Scholar, M. Biol. 2003; PubMed Scopus Google Scholar). Similarly, the cholesterol in the plasma membranes of cells appears to a level that the phospholipids (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar, 20Lange Y. Ye J. Rigney M. Steck T.L. J. Lipid Res. 1999; 40: 2264-2270Abstract Full Text Full Text PDF PubMed Google Scholar). the activity of the cholesterol in the plasma membrane the physiological threshold, the excess is to both to plasma and to membranes and to cholesterol in the endoplasmic reticulum then the regulatory elements to the abundance of cell cholesterol through its the of its and the of the of for its (8Lange Y. Ye J. Steck T.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11664-11667Crossref PubMed Scopus (128) Google Scholar). the from the of the plasma membrane bilayer, are by a of Similarly, in the case of membrane intercalators that displace cholesterol from its association with plasma membrane bilayer the high chemical activity of the cholesterol also increase the pool in the endoplasmic This is for intercalators the of activity in is that, by this certain membrane intercalators could be used to in We London of for